Roles of metabotropic glutamate receptor 5 in low [Mg2+]o-induced interictal epileptiform activity in rat hippocampal slices.

Group I metabotropic glutamate receptors (mGluRs) modulate postsynaptic neuronal excitability and epileptogenesis. We investigated roles of group I mGluRs on low extracellular Mg2+ concentration ([Mg2+]o)-induced epileptiform activity and neuronal cell death in the CA1 regions of isolated rat hippocampal slices without the entorhinal cortex using extracellular recording and propidium iodide staining. Exposure to Mg2+-free artificial cerebrospinal fluid can induce interictal epileptiform activity in the CA1 regions of rat hippocampal slices. MPEP, a mGluR 5 antagonist, significantly inhibited the spike firing of the low [Mg2+]o-induced epileptiform activity, whereas LY367385, a mGluR1 antagonist, did not. DHPG, a group 1 mGluR agonist, significantly increased the spike firing of the epileptiform activity. U73122, a PLC inhibitor, inhibited the spike firing. Thapsigargin, an ER Ca2+-ATPase antagonist, significantly inhibited the spike firing and amplitude of the epileptiform activity. Both the IP3 receptor antagonist 2-APB and the ryanodine receptor antagonist dantrolene significantly inhibited the spike firing. The PKC inhibitors such as chelerythrine and GF109203X, significantly increased the spike firing. Flufenamic acid, a relatively specific TRPC 1, 4, 5 channel antagonist, significantly inhibited the spike firing, whereas SKF96365, a relatively non-specific TRPC channel antagonist, did not. MPEP significantly decreased low [Mg2+]o DMEM-induced neuronal cell death in the CA1 regions, but LY367385 did not. We suggest that mGluR 5 is involved in low [Mg2+]oinduced interictal epileptiform activity in the CA1 regions of rat hippocampal slices through PLC, release of Ca2+ from intracellular stores and PKC and TRPC channels, which could be involved in neuronal cell death.

Atypical antidepressant mirtazapine inhibits 5-hydroxytryptamine3 receptor currents in NCB-20 cells.

Mirtazapine, an atypical antidepressant, is known to enhance serotonergic transmission by inhibiting the 5-hydroxytryptamine (5-HT)1A, 5-HT2C, and 5-HT3 receptors. However, the mechanism of action on the 5-HT3 receptor remains unclear. We investigated the inhibitory mechanisms of mirtazapine on 5-HT3 receptors of NCB20 neuroblastoma cells using the whole-cell voltage-clamp method. Mirtazapine inhibited the 5-HT3 receptor currents in a concentration-dependent manner, and the inhibitory effect was influenced by the concentration of 5-HT. When mirtazapine was co-applied to 5-HT, the maximal response of the 5-HT3 receptor current was reduced and EC50 was increased, suggesting that mirtazapine might act as a non-competitive inhibitor. Inhibition of 5-HT3 current by mirtazapine was stronger in pre-application than in co-application, which suggests that mirtazapine might act as a closed state inhibitor. This finding was further supported by no use-dependency of the mirtazapine for 5-HT3 receptor inhibition. Finally, mirtazapine accelerated the desensitization and deactivation process in a concentration-dependent manner. The difference in recovery time showed that mirtazapine drastically influences the desensitization process than the deactivation process. These mechanistic characteristics of mirtazapine support the understanding of the relationship between the 5-HT3 receptor and atypical antidepressants.

Selective serotonin reuptake inhibitor escitalopram inhibits 5-HT3 receptor currents in NCB-20 cells.

Escitalopram is one of selective serotonin reuptake inhibitor antidepressants. As an S-enantiomer of citalopram, it shows better therapeutic outcome in depression and anxiety disorder treatment because it has higher selectivity for serotonin reuptake transporter than citalopram. The objective of this study was to determine the direct inhibitory effect of escitalopram on 5-hydroxytryptamine type 3 (5-HT3) receptor currents and study its blocking mechanism to explore additional pharmacological effects of escitalopram through 5-HT3 receptors. Using a whole-cell voltage clamp method, we recorded currents of 5-HT3 receptors when 5-HT was applied alone or co-applied with escitalopram in cultured NCB-20 neuroblastoma cells known to express 5-HT3 receptors. 5-HT induced currents were inhibited by escitalopram in a concentration-dependent manner. EC50 of 5-HT on 5-HT3 receptor currents was increased by escitalopram while the maximal peak amplitude was reduced by escitalopram. The inhibitory effect of escitalopram was voltage independent. Escitalopram worked more effectively when it was co-applied with 5-HT than pre-application of escitalopram. Moreover, escitalopram showed fast association and dissociation to the open state of 5-HT3 receptor channel with accelerating receptor desensitization. Although escitalopram accelerated 5-HT3 receptor desensitization, it did not change the time course of desensitization recovery. These results suggest that escitalopram can inhibit 5-HT3 receptor currents in a non-competitive manner with the mechanism of open channel blocking.

Gastroprokinetic agent, mosapride inhibits 5-HT3 receptor currents in NCB-20 cells.

Mosapride accelerates gastric emptying by acting on 5-hydroxytryptamine type 4 (5-HT4) receptor and is frequently used in the treatment of gastrointestinal (GI) disorders requiring gastroprokinetic efficacy. We tested the effect of mosapride on 5-hydroxytryptamine type 3 (5-HT3) receptor currents because the 5-HT3 receptors are also known to be expressed in the GI system and have an important role in the regulation of GI functions. Using the whole-cell voltage clamp method, we compared the currents of the 5-HT3 receptors when 5-HT was applied alone or was co-applied with mosapride in cultured NCB-20 cells known to express 5-HT3 receptors. The 5-HT3 receptor current amplitudes were inhibited by mosapride in a concentration-dependent manner. Mosapride blocked the peak currents evoked by the application of 5-HT in a competitive manner because the EC50 shifted to the right without changing the maximal effect. The rise slopes of 5-HT3 receptor currents were decreased by mosapride. Pre-application of mosapride before co-application, augmented the inhibitory effect of mosapride, which suggests a closed channel blocking mechanism. Mosapride also blocked the opened 5-HT3 receptor because it inhibited the 5-HT3 receptor current in the middle of the application of 5-HT. It accelerated desensitization of the 5-HT3 receptor but did not change the recovery process from the receptor desensitization. There were no voltage-, or use-dependency in its blocking effects. These results suggest that mosapride inhibited the 5-HT3 receptor through a competitive blocking mechanism probably by binding to the receptor in closed state, which could be involved in the pharmacological effects of mosapride to treat GI disorders.

Tricyclic antidepressant amitriptyline inhibits 5-hydroxytryptamine 3 receptor currents in NCB-20 cells.

Amitriptyline, a tricyclic antidepressant, is commonly used to treat depression and neuropathic pain, but its mechanism is still unclear. We tested the effect of amitriptyline on 5-hydroxytryptamine 3 (5-HT3) receptor currents and studied its blocking mechanism because the clinical applications of amitriptyline overlapped with 5-HT3 receptor therapeutic potentials. Using a whole-cell voltage clamp method, we recorded the currents of the 5-HT3 receptor when 5-HT was applied alone or co-applied with amitriptyline in cultured NCB-20 neuroblastoma cells known to express 5-HT3 receptors. To elucidate the mechanism of amitriptyline, we simulated the 5-HT3 receptor currents using Berkeley Madonna® software and calculated the rate constants of the agonist binding and receptor transition steps. The 5-HT3 receptor currents were inhibited by amitriptyline in a concentration-dependent, voltage-independent manner, and a competitive mode. Amitriptyline accelerated the desensitization of the 5-HT3 receptor. When amitriptyline was applied before 5-HT treatment, the currents rose slowly until the end of 5-HT treatment. When amitriptyline was co-applied with 5-HT, currents rose and decayed rapidly. Peak current amplitudes were decreased in both applications. All macroscopic currents recorded in whole cell voltage clamping experiments were reproduced by simulation and the changes of rate constants by amitriptyline were correlated with macroscopic current recording data. These results suggest that amitriptyline blocks the 5-HT3 receptor by close and open state blocking mechanisms, in a competitive manner. We could expand an understanding of pharmacological mechanisms of amitriptyline related to the modulation of a 5-HT3 receptor, a potential target of neurologic and psychiatric diseases through this study.

Lamotrigine, an antiepileptic drug, inhibits 5-HT3 receptor currents in NCB-20 neuroblastoma cells.

Lamotrigine is an antiepileptic drug widely used to treat epileptic seizures. Using whole-cell voltage clamp recordings in combination with a fast drug application approach, we investigated the effects of lamotrigine on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells. Co-application of lamotrigine (1~300 µM) resulted in a concentration-dependent reduction in peak amplitude of currents induced by 3 µM of 5-HT for an IC50 value of 28.2±3.6 µM with a Hill coefficient of 1.2±0.1. These peak amplitude decreases were accompanied by the rise slope reduction. In addition, 5-HT3-mediated currents evoked by 1 mM dopamine, a partial 5-HT3 receptor agonist, were inhibited by lamotrigine co-application. The EC50 of 5-HT for 5-HT3 receptor currents were shifted to the right by co-application of lamotrigine without a significant change of maximal effect. Currents activated by 5-HT and lamotrigine co-application in the presence of 1 min pretreatment of lamotrigine were similar to those activated by 5-HT and lamotrigine co-application alone. Moreover, subsequent application of lamotrigine in the presence of 5-HT and 5-hydroxyindole, known to attenuate 5-HT3 receptor desensitization, inhibited 5-HT3 receptor currents in a concentration-dependent manner. The deactivation of 5-HT3 receptor was delayed by washing with an external solution containing lamotrigine. Lamotrigine accelerated the desensitization process of 5-HT3 receptors. There was no voltage-dependency in the inhibitory effects of lamotrigine on the 5-HT3 receptor currents. These results indicate that lamotrigine inhibits 5-HT3-activated currents in a competitive manner by binding to the open state of the channels and blocking channel activation or accelerating receptor desensitization.

Acepromazine inhibits hERG potassium ion channels expressed in human embryonic kidney 293 cells.

The effects of acepromazine on human ether-à-go-go-related gene (hERG) potassium channels were investigated using whole-cell voltage-clamp technique in human embryonic kidney (HEK293) cells transfected with hERG. The hERG currents were recorded with or without acepromazine, and the steady-state and peak tail currents were analyzed for the evaluating the drug effects. Acepromazine inhibited the hERG currents in a concentration-dependent manner with an IC50 value of 1.5 µM and Hill coefficient of 1.1. Acepromazine blocked hERG currents in a voltage-dependent manner between -40 and +10 mV. Before and after application of acepromazine, the half activation potentials of hERG currents changed to hyperpolarizing direction. Acepromazine blocked both the steady-state hERG currents by depolarizing pulse and the peak tail currents by repolarizing pulse; however, the extent of blocking by acepromazine in the repolarizing pulse was more profound than that in the depolarizing pulse, indicating that acepromazine has a high affinity for the open state of the channels, with a relatively lower affinity for the closed state of hERG channels. A fast application of acepromazine during the tail currents inhibited the open state of hERG channels in a concentration-dependent. The steady-state inactivation of hERG currents shifted to the hyperpolarized direction by acepromazine. These results suggest that acepromazine inhibits the hERG channels probably by an open- and inactivated-channel blocking mechanism. Regarding to the fact that the hERG channels are the potential target of drug-induced long QT syndrome, our results suggest that acepromazine can possibly induce a cardiac arrhythmia through the inhibition of hERG channels.

Endoxifen, the active metabolite of tamoxifen, inhibits cloned hERG potassium channels.

The effects of tamoxifen, and its active metabolite endoxifen (4-hydroxy-N-desmethyl-tamoxifen), on hERG currents stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and an immunoblot assay. Tamoxifen and endoxifen inhibited hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 1.2 and 1.6µM, respectively. The steady-state activation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of endoxifen. The voltage-dependent inhibition of hERG currents by endoxifen increased steeply in the voltage range of channel activation. The inhibition by endoxifen displayed a shallow voltage dependence (δ=0.18) in the full activation voltage range. A fast application of endoxifen induced a reversible block of hERG tail currents during repolarization in a concentration-dependent manner, which suggested an interaction with the open state of the channel. Endoxifen also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an interaction with the activated (open and/or inactivated) states of the channels. Tamoxifen and endoxifen inhibited the hERG channel protein trafficking to the plasma membrane in a concentration-dependent manner with endoxifen being more potent than tamoxifen. These results indicated that tamoxifen and endoxifen inhibited the hERG current by direct channel blockage and by the disruption of channel trafficking to the plasma membrane in a concentration-dependent manner. A therapeutic concentration of endoxifen inhibited the hERG current by preferentially interacting with the activated (open and/or inactivated) states of the channel.

Effects of donepezil on hERG potassium channels.

Donepezil is a potent, selective inhibitor of acetylcholinesterase, which is used for the treatment of Alzheimer's disease. Whole-cell patch-clamp technique and Western blot analyses were used to study the effects of donepezil on the human ether-a-go-go-related gene (hERG) channel. Donepezil inhibited the tail current of the hERG in a concentration-dependent manner with an IC50 of 1.3 µM. The metabolites of donepezil, 6-ODD and 5-ODD, inhibited the hERG currents in a similar concentration-dependent manner; the IC50 values were 1.0 and 1.5 µM, respectively. A fast drug perfusion system demonstrated that donepezil interacted with both the open and inactivated states of the hERG. A fast application of donepezil during the tail currents inhibited the open state of the hERG in a concentration-dependent manner with an IC50 of 2.7 µM. Kinetic analysis of donepezil in an open state of the hERG yielded blocking and unblocking rate constants of 0.54 µM(-1)s(-1) and 1.82 s(-1), respectively. The block of the hERG by donepezil was voltage-dependent with a steep increase across the voltage range of channel activation. Donepezil caused a reduction in the hERG channel protein trafficking to the plasma membrane at low concentration, but decreased the channel protein expression at higher concentrations. These results suggest that donepezil inhibited the hERG at a supratherapeutic concentration, and that it did so by preferentially binding to the activated (open and/or inactivated) states of the channels and by inhibiting the trafficking and expression of the hERG channel protein in the plasma membrane.

Raloxifene inhibits cloned Kv4.3 channels in an estrogen receptor-independent manner.

Raloxifene is widely used for the treatment and prevention of postmenopausal osteoporosis. We examined the effects of raloxifene on the Kv4.3 currents expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique and on the long-term modulation of Kv4.3 messenger RNA (mRNA) by real-time PCR analysis. Raloxifene decreased the Kv4.3 currents with an IC50 of 2.0 µM and accelerated the inactivation and activation kinetics in a concentration-dependent manner. The inhibitory effects of raloxifene on Kv4.3 were time-dependent: the association and dissociation rate constants for raloxifene were 9.5 µM(-1) s(-1) and 23.0 s(-1), respectively. The inhibition by raloxifene was voltage-dependent (δ = 0.13). Raloxifene shifted the steady-state inactivation curves in a hyperpolarizing direction and accelerated the closed-state inactivation of Kv4.3. Raloxifene slowed the time course of recovery from inactivation, thus producing a use-dependent inhibition of Kv4.3. ß-Estradiol and tamoxifen had little effect on Kv4.3. A preincubation of ICI 182,780, an estrogen receptor antagonist, for 1 h had no effect on the inhibitory effect of raloxifene on Kv4.3. The metabolites of raloxifene, raloxifene-4'-glucuronide and raloxifene-6'-glucuronide, had little or no effect on Kv4.3. Coexpression of KChIP2 subunits did not alter the drug potency and steady-state inactivation of Kv4.3 channels. Long-term exposure to raloxifene (24 h) significantly decreased the expression level of Kv4.3 mRNA. This effect was not abolished by the coincubation with ICI 182,780. Raloxifene inhibited Kv4.3 channels by interacting with their open state during depolarization and with the closed state at subthreshold potentials. This effect was not mediated via an estrogen receptor.

Effects of haloperidol on Kv4.3 potassium channels.

Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 µM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials.

Escitalopram block of hERG potassium channels.

Escitalopram, a selective serotonin reuptake inhibitor, is the pharmacologically active S-enantiomer of the racemic mixture of RS-citalopram and is widely used in the treatment of depression. The effects of escitalopram and citalopram on the human ether-a-go-go-related gene (hERG) channels expressed in human embryonic kidney cells were investigated using voltage-clamp and Western blot analyses. Both drugs blocked hERG currents in a concentration-dependent manner with an IC50 value of 2.6 µM for escitalopram and an IC50 value of 3.2 µM for citalopram. The blocking of hERG by escitalopram was voltage-dependent, with a steep increase across the voltage range of channel activation. However, voltage independence was observed over the full range of activation. The blocking by escitalopram was frequency dependent. A rapid application of escitalopram induced a rapid and reversible blocking of the tail current of hERG. The extent of the blocking by escitalopram during the depolarizing pulse was less than that during the repolarizing pulse, suggesting that escitalopram has a high affinity for the open state of the hERG channel, with a relatively lower affinity for the inactivated state. Both escitalopram and citalopram produced a reduction of hERG channel protein trafficking to the plasma membrane but did not affect the short-term internalization of the hERG channel. These results suggest that escitalopram blocked hERG currents at a supratherapeutic concentration and that it did so by preferentially binding to both the open and the inactivated states of the channels and by inhibiting the trafficking of hERG channel protein to the plasma membrane.

Effect of mosapride on Kv4.3 potassium channels expressed in CHO cells.

Mosapride and cisapride are gastroprokinetic agents with 5-hydroxytryptamine4 receptor agonist activity and have been widely used in the treatment of a variety of gastrointestinal disorders. The effects of mosapride and cisapride on cloned Kv4.3 channels stably expressed in Chinese hamster ovary cells were investigated using the whole-cell patch-clamp technique. Mosapride and cisapride inhibited Kv4.3 in a concentration-dependent manner with IC50 values of 15.2 and 9.8 µM, respectively. Mosapride accelerated the rate of inactivation and activation of Kv4.3 in a concentration-dependent manner and thereby decreased the time to peak. The rate constants of association (k +1) and dissociation (k -1) for mosapride were 9.9 µM(-1) s(-1) and 151.3 s(-1), respectively. The K D (k -1/k +1) was 16.2 µM, similar to the IC50 value calculated from the concentration-response curve. Voltage-dependent inhibition by mosapride was observed in the voltage range for channel opening but was not observed over a voltage range in which all Kv4.3 channels were open. Both the steady-state activation and inactivation curves of Kv4.3 were shifted in the hyperpolarizing direction in the presence of mosapride. Mosapride also caused a substantial acceleration in closed-state inactivation of Kv4.3. Mosapride produced use-dependent inhibition, which was consistent with a slow recovery from inactivation of Kv4.3. M1 and norcisapride, the major metabolites of mosapride and cisapride, respectively, had little or no effect on Kv4.3. These results indicate that mosapride inhibits Kv4.3 by both preferential binding to the open state of the channels during depolarization and acceleration of the closed-state inactivation at subthreshold potentials.

Fluoxetine suppresses synaptically induced [Ca²âº]i spikes and excitotoxicity in cultured rat hippocampal neurons.

Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca(2+) channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca(2+)](i) spikes. We investigated the effects of fluoxetine on [Ca(2+)](i) spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg(2+) in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca(2+)](i) spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5 min inhibited [Ca(2+)](i) increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca(2+)](i) responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca(2+)](i) responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg(2+)](o)-induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca(2+)](i) signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca(2+)](i) spikes and neuronal cell death.

Involvement of the endocannabinoid system in ethanol-induced corticostriatal synaptic depression.

Ethanol is a wildly abused substance that causes various problems and damage in our society. We examined the connection between the action of ethanol and the endocannabinoid system in corticostriatal synaptic transmission by recording excitatory post-synaptic currents (EPSCs). Acute treatment of ethanol (100 mM) inhibited corticostriatal EPSCs. In the presence of AM 251 (5 µM), a cannabinoid 1 (CB(1))-receptor antagonist, or AM 404 (5 µM), a cannabinoid transporter inhibitor, the inhibition of corticostriatal EPSCs caused by ethanol was significantly reduced. This result suggests the possibility that the endocannabinoid system is involved in the action of ethanol. To support this result, brain slices were pre-treated with WIN 55,212-2 (1 µM), a CB(1)-receptor agonist, following treatment of ethanol or treated with WIN 55,212-2 alone. There was no significant difference between each other, indicating that when CB(1) receptors are previously activated, the effect of ethanol is blunted. These results suggest that the activation of the endocannabinoid system is one of the possible mechanisms involved in ethanol-induced corticostriatal synaptic depression.

Effects of grape seed proanthocyanidin on 5-hydroxytryptamine(3) receptors in NCB-20 neuroblastoma cells.

Proanthocyanidin is a phenolic compound present in plants, that has antioxidant, antinociceptive, anti-emetic, and neuroprotective properties. We investigated the actions of proanthocyanidin from grape seeds on 5-hydroxytryptamine (5-HT)(3) receptors in NCB-20 neuroblastoma cells using a whole-cell voltage clamp technique. Co-treatment of proanthocyanidin (0.3-100 µg/ml) and 3 µM 5-HT (near EC(50)) produced a slight inhibition of 5-HT-induced inward peak current (I(5-HT)) in NCB-20 cells, but pretreatment with proanthocyanidin for 30 s before application of 5-HT induced a much larger inhibition of I(5-HT) in an irreversible, concentration- and time-dependent manner (IC(50)=6.5±0.4 µg/ml, Hill coefficient=2.5±0.1). Proanthocyanidin also produced a concentration-dependent inhibition of currents induced by 30 µM 5-HT, near-maximal concentration (IC(50)=22.1±0.4 µg/ml, Hill coefficient=2.4±0.1). High concentrations (≧30 µg/ml) of proanthocyanidin caused a concentration-dependent inhibition of the activation and desensitization of currents induced by 30 µM 5-HT. Further studies showed that pretreatment of 20 µg/ml proanthocyanidin caused not only a rightward shift of the dose-response curve for 5-HT (EC(50) shift from 2.7±0.4 to 6.2±0.5 µM), but also a decreased E(max) (inhibition by 37.5±1.3%). The proanthocyanidin-induced inhibition of 5-HT(3) receptors did not show a significant difference within the testing holding potential ranges (-50-+30 mV). These results suggest that proanthocyanidin inhibits 5-HT(3) receptor function in NCB-20 cells in a noncompetitive mode, and that this inhibitory effect of proanthocyanidin probably contributes to the pharmacological actions of proanthocyanidin.

Electrophysiological Characterization of AMPA and NMDA Receptors in Rat Dorsal Striatum.

The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20+/-0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26+/-0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32+/-0.03. The rectification index (control 2.39+/-0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02+/-0.11 and 0.93+/-0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.

Orexin-A increases cell surface expression of AMPA receptors in the striatum.

Accumulating evidence suggests that orexin signaling is involved in reward and motivation circuit functions. However, the underlying mechanisms are not yet fully understood. Here, we show that orexin-A potentiates AMPAR-mediated synaptic transmission in the striatum, possibly by regulating the surface expression of AMPARs. Primary culture of striatal neurons revealed increased surface expression of AMPARs following orexin-A treatment. The increase in surface-expressed AMPARs induced by orexin-A treatment was dependent on both ERK activation and the presence of extracellular Ca(2+). In the corticostriatal synapses of rat brain slices, orexin-A bath-application caused a delayed increase in the AMPAR/NMDAR EPSC ratio, suggesting that orexin-A sets in motion a series of events that lead to functional alterations in the striatal circuits. Our findings provide a potential link between the activation of orexin signaling in the striatum in response to addictive substances and neural adaptations in the reward circuitry that may mediate the long-lasting addiction-related behaviors.

Direct effects of riluzole on 5-hydroxytryptamine (5-HT)3 receptor-activated ion currents in NCB-20 neuroblastoma cells.

The pharmacological action of riluzole, a drug that has been approved as a neuroprotective agent for the treatment of amyotrophic lateral sclerosis, has not yet been established. We examined the effects of riluzole on 5-hydroxytryptamine (5-HT)3) receptors in NCB-20 neuroblastoma cells using the whole-cell voltage clamp technique combined with a fast drug application method. Co-application of riluzole (1 - 300 microM, 5 s) produced a dose-dependent reduction in peak amplitudes and in the rise slope of the currents induced by 2 microM 5-HT. In addition, 5-HT3-mediated currents evoked by dopamine, a partial 5-HT3-receptor agonist, were inhibited by riluzole co-application. These inhibitory effects were clearly shown at low concentrations of 5-HT. The decay time constants of the receptor desensitization and deactivation were also significantly attenuated by riluzole. G-protein inhibitors (pertussis toxin and guanosine 5'-[beta-thio] diphosphate) did not completely block these inhibitory actions of riluzole. These results indicate that riluzole inhibits 5-HT3-induced ion currents directly by slowing channel activation in NCB-20 neuroblastoma cells.

Effects of Apigenin on Glutamate-induced [Ca](i) Increases in Cultured Rat Hippocampal Neurons.

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin (1 microM to 100 microM) for 5 min inhibited glutamate (100 microM, 1 min) induced [Ca(2+)](i) increase, concentration-dependently. Pretreatment with apigenin (30 microM) for 5 min significantly decreased the [Ca(2+)](i) responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 microM, 1 min) and N-methyl-D-aspartate (NMDA, 100 microM, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca(2+)](i) response induced by 50 mM KCl solution, decreased the [Ca(2+)](i) responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100 microM, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca(2+)](i) responses. Furthermore, treatment with apigenin (30 microM) significantly inhibited the amplitude and frequency of 0.1 mM [Mg(2+)](o)-induced [Ca(2+)](i) spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.