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1.
J Neurochem ; 128(6): 927-37, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24164423

ABSTRACT

Because the cholinergic system is down-regulated in the brain of Alzheimer's disease patients, cognitive deficits in Alzheimer's disease patients are significantly improved by rivastigmine treatment. To address the mechanism underlying rivastigmine-induced memory improvements, we chronically treated olfactory bulbectomized (OBX) mice with rivastigmine. The chronic rivastigmine treatments for 12-13 days starting at 10 days after OBX operation significantly improved memory-related behaviors assessed by Y-maze task, novel object recognition task, passive avoidance task, and Barnes maze task, whereas the single rivastigmine treatment failed to improve the memory. Consistent with the improved memory-related behaviors, long-term potentiation in the hippocampal CA1 region was markedly restored by rivastigmine treatments. In immunoblotting analyses, the reductions of calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in the CA1 region in OBX mice were significantly restored by rivastigmine treatments. In addition, phosphorylation of AMPAR subunit glutamate receptor 1 (GluA1) (Ser-831) and cAMP-responsive element-binding protein (Ser-133) as downstream targets of CaMKII and CaMKIV, respectively, in the CA1 region was also significantly restored by chronic rivastigmine treatments. Finally, we confirmed that rivastigmine-induced improvements of memory-related behaviors and long-term potentiation were not obtained in CaMKIIα(+/-) mice. On the other hand, CaMKIV(-/-) mice did not exhibit the cognitive impairments. Taken together, the stimulation of CaMKII activity in the hippocampus is essential for rivastigmine-induced memory improvement in OBX mice.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 4/metabolism , Memory Disorders/metabolism , Memory/physiology , Phenylcarbamates/pharmacology , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Animals , Animals, Outbred Strains , CA1 Region, Hippocampal/drug effects , CA1 Region, Hippocampal/physiology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 4/genetics , Cholinesterase Inhibitors/pharmacology , Denervation/methods , Disease Models, Animal , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Memory/drug effects , Memory Disorders/drug therapy , Mice , Olfactory Bulb/surgery , Rivastigmine
2.
Hippocampus ; 23(10): 942-51, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23733502

ABSTRACT

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 µM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.


Subject(s)
CA1 Region, Hippocampal/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Glycine , Kynurenic Acid/pharmacology , Long-Term Potentiation/drug effects , Nootropic Agents/pharmacology , Piperazines/pharmacology , Receptors, N-Methyl-D-Aspartate/drug effects , Synaptic Transmission/drug effects , Animals , CA1 Region, Hippocampal/surgery , Dose-Response Relationship, Drug , Electrodes, Implanted , Excitatory Amino Acid Antagonists/administration & dosage , Glycine/metabolism , Kynurenic Acid/administration & dosage , Male , Mice , Mice, Inbred C57BL , Nootropic Agents/administration & dosage , Piperazines/administration & dosage , Piperidines/administration & dosage , Piperidines/pharmacology , Polyamines/metabolism , Protein Binding/physiology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors
3.
Behav Brain Res ; 242: 150-7, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23295391

ABSTRACT

Alzheimer's disease (AD) shows degeneration of the cholinergic system in the medial septum, thereby eliciting down-regulation of the olfactory function in patients. We have previously reported that olfactory bulbectomized (OBX) mice show hippocampus-dependent memory impairment as assessed by memory-related behavioral tasks and hippocampal long-term potentiation (LTP). In the present study, we focused whether novel pyrrolidone nootropic drug sunifiram improves both memory impairment and depression observed in OBX mice. OBX mice were administered once a day for 7-12 days with sunifiram (0.01-1.0mg/kg p.o.) from 10 days after operation with or without gavestinel (10mg/kg i.p.), which is glycine-binding site inhibitor of N-methyl-d-aspartate receptor (NMDAR). The spatial reference memory assessed by Y-maze and short-term memory assessed by novel object recognition task were significantly improved by sunifiram treatment in OBX mice. Sunifiram also restored hippocampal LTP injured in OBX mice without treatment with gavestinel. By contrast, sunifiram treatment did not ameliorate the depressive behaviors assessed by tail suspension task in OBX mice. Notably, sunifiram treatment restored CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA1 region from OBX mice to the levels of control mice. Likewise, sunifiram treatment improved PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) phosphorylation to the control levels. Stimulation of CaMKII and PKC autophosphorylation by sunifiram was significantly inhibited by pre-treatment with gavestinel. However, sunifiram treatment did not affect the phosphorylation of CaMKIV (Thr-196) and ERK. Taken together, sunifiram ameliorates OBX-induced deficits of memory-related behaviors and impaired LTP in the hippocampal CA1 region via stimulation of glycine-binding site of NMDAR.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cognition Disorders/drug therapy , Nootropic Agents/therapeutic use , Piperazines/therapeutic use , Protein Kinase C/metabolism , Analysis of Variance , Animals , Cognition Disorders/etiology , Cognition Disorders/pathology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Excitatory Amino Acid Antagonists/administration & dosage , Excitatory Postsynaptic Potentials/drug effects , Exploratory Behavior/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Hindlimb Suspension , Hippocampus/cytology , In Vitro Techniques , Indoles/administration & dosage , Long-Term Potentiation/drug effects , Male , Maze Learning/drug effects , Mice , Neurons/drug effects , Olfactory Bulb/surgery , Phosphorylation , Receptors, Glutamate/metabolism , Recognition, Psychology/drug effects , Tubulin/metabolism
4.
Neuroreport ; 23(10): 601-5, 2012 Jul 11.
Article in English | MEDLINE | ID: mdl-22664778

ABSTRACT

The purinergic P2X4 receptors (P2X4Rs) of spinal microglia are upregulated after a peripheral nerve injury and play important roles in the pathogenesis of chronic pain. The effects of general anesthetics on chronic pain and the mechanisms are still unclear. The aim of this study is to examine the effects of general anesthetics on microglial P2X4Rs. Currents induced by ATP were recorded by the whole-cell clamp technique using a mouse microglial cell line (MG5). Isoflurane and sevoflurane, ketamine, thiopental, midazolam, and propofol were coapplied with ATP using the U-tube system or added to the external perfusate. ATP-induced two distinct types of current: P2X4R-mediated and P2X7R-mediated currents. P2X4R-mediated currents were identified pharmacologically and isolated. Volatile anesthetics including sevoflurane and isoflurane and intravenous anesthetics including thiopental, ketamine, and midazolam had no effect at clinically relevant concentrations (n=5-8). Propofol showed a dual effect, potentiating at lower concentrations (0.3-3 µM) and inhibiting at higher concentrations (IC50 57 µM). The maximum enhancement was observed at 1 µM propofol (143±5% of control, n=5). Propofol (1 µM) shifted the dose-response curve for the P2X4R currents to lower concentrations of ATP and increased the maximum amplitude. Propofol exerted dual actions on P2X4R-mediated currents at clinically relevant concentrations. This may suggest that the administration of propofol could affect the development of chronic pain through the modulation of microglial P2X4R responses.


Subject(s)
Anesthetics, General/pharmacology , Microglia/drug effects , Microglia/physiology , Receptors, Purinergic P2X4/physiology , Animals , Cell Line , Mice , Propofol/pharmacology , Purinergic P2X Receptor Agonists/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology
5.
Brain Res ; 1435: 15-23, 2012 Jan 30.
Article in English | MEDLINE | ID: mdl-22177663

ABSTRACT

Proton channels are gated by voltage and pH gradients, and play an important role in the microglial production of pro-inflammatory cytokines, which are known to be suppressed by antidepressants. In the present study we tested the hypothesis that cytokine inhibition by antidepressants is due to an inhibitory action on proton currents by comparing their effects on tumor necrosis factor-α production with the effects on the proton currents in BV2 murine microglial cells. Imipramine, amitriptyline, desipramine and fluoxetine potently and reversibly inhibited proton currents at micromolar concentrations at an intracellular/extracellular pH gradient of 5.5/7.3. Raising extracellular pH to 8.3 sped up the rate and enhanced the extent of block whereas raising intracellular pH to 6.3 reduced the blocking potency of imipramine. These results support a mechanism where the uncharged drug form penetrates the cell membrane, and the charged form blocks the proton channel from the internal side of membrane. This mode of action was corroborated by an experiment with imipraminium, a permanently charged quaternary derivative, which showed far less block compared to imipramine. The lipopolysaccharide-induced release of tumor necrosis factor-α was inhibited by imipramine at concentrations comparable to those inhibiting the proton current. These results support the hypothesis that tumor necrosis factor-α inhibition by imipramine is related to its inhibitory effects on proton channels.


Subject(s)
Antidepressive Agents/pharmacology , Membrane Potentials/drug effects , Microglia/drug effects , Protons , Tumor Necrosis Factor-alpha/metabolism , Analysis of Variance , Animals , Biophysics , Cell Line, Transformed , Cytokines/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Hydrogen-Ion Concentration , Lipopolysaccharides/pharmacology , Membrane Potentials/physiology , Mice
6.
J Physiol ; 590(4): 827-44, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22183729

ABSTRACT

Voltage-gated proton channels play crucial roles during the respiratory burst in phagocytes, such as microglia. As local anaesthetics have a variety of anti-inflammatory properties, including inhibition of phagocytosis, they may act on the proton channels. Most local anaesthetics are tertiary amines and may affect proton channels through modification of pH(i) as weak bases. To test these hypotheses, the effects of lidocaine and bupivacaine on proton channels were examined in a rat microglial cell line (GMI-R1) as a function of pH(o) and pH(i). Both lidocaine and bupivacaine reversibly decreased the current, with IC(50) values of ∼1.2 and ∼0.5 mM, respectively, at pH(o)/pH(i) 7.3/5.5. The inhibition was enhanced with either pH(o) increase or pH(i) decrease, suggesting that the protonation of the base forms inside the cell contributed to the inhibitory effects. Both local anaesthetics shifted the reversal potentials to more positive voltages, indicating increases in pH(i). The potencies of inhibition were correlated well with the degree of increase in pH(i). The lidocaine-induced inhibition was eliminated when the pH(i) increases were cancelled by co-application of a weak acid, butyrate. The cytosolic alkalizations by lidocaine and bupivacaine were confirmed using a pH-sensitive fluorescent dye, BCECF, in non-voltage-clamped cells. Furthermore, chemiluminescence measurement proved that both anaesthetics inhibited production of reactive oxygen species by the cells. In conclusion, lidocaine and bupivacaine inhibit proton channels primarily by the weak base mechanism via an increase in pH(i). This is a novel mechanism underlying actions of local anaesthtics.


Subject(s)
Anesthetics, Local/pharmacology , Bupivacaine/pharmacology , Ion Channels/drug effects , Lidocaine/pharmacology , Protons , Animals , Cell Line , Hydrogen-Ion Concentration , Ion Channels/physiology , Microglia/drug effects , Microglia/physiology , Rats , Reactive Oxygen Species/metabolism
7.
Pestic Biochem Physiol ; 97(2): 149-152, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20563240

ABSTRACT

Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABA(A) receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC(50)s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABA(A) receptors. Moreover, GluCls of cockroach neurons had low IC(50)s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC(50)s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.

8.
J Neurochem ; 110(1): 170-81, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19457128

ABSTRACT

Aberrant behaviors related to learning and memory in olfactory bulbectomized (OBX) mice have been documented in the previous studies. We reported that the impairment of long-term potentiation (LTP) of hippocampal CA1 regions from OBX mice was associated with down-regulation of CaM kinase II (CaMKII) and protein kinase C (PKC) activities. We now demonstrated that the nootropic drug, nefiracetam, significantly improved spatial reference memory-related behaviors as assessed by Y-maze and novel object recognition task in OBX mice. Nefiracetam also restored hippocampal LTP injured in OBX mice. Nefiracetam treatment restored LTP-induced PKCalpha (Ser657) and NR1 (Ser896) phosphorylation as well as increase in their basal phosphorylation in the hippocampal CA1 region of OBX mice. Likewise, nefiracetam improved LTP-induced CaMKIIalpha (Thr286) autophosphorylation and GluR1 (Ser831) phosphorylation and increased their basal phosphorylation. The enhancement of PKCalpha (Ser657) and CaMKIIalpha (Thr286) autophosphorylation by nefiracetam was inhibited by treatment with (+/-)-alpha-Methyl-(4-carboxyphenyl)glycine and DL-2-Amino-5-phosphonovaleric acid, respectively. The enhancement of LTP induced by nefiracetam is inhibited by treatment with 2-methyl-6-(phenylethynyl)-pyridine, but not by treatment with LY367385, suggesting that metabotropic glutamate receptor 5 (mGluR5) but not mGluR1 is involved in the nefiracetam-induced LTP enhancement. Taken together, nefiracetam ameliorates OBX-induced deficits in memory-related behaviors and impairment of LTP in the hippocampal CA1 region through activation of NMDAR and mGluR5, thereby leading to an increase in activities of CaMKIIalpha (Thr286) and PKCalpha (Ser657), respectively.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/drug effects , Hippocampus/drug effects , Memory Disorders/drug therapy , Protein Kinase C/drug effects , Pyrrolidinones/pharmacology , Receptors, Glutamate/drug effects , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Denervation , Disease Models, Animal , Enzyme Activation/drug effects , Enzyme Activation/physiology , Hippocampus/metabolism , Hippocampus/physiopathology , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Memory Disorders/metabolism , Memory Disorders/physiopathology , Mice , Nootropic Agents/pharmacology , Nootropic Agents/therapeutic use , Olfactory Bulb/injuries , Olfactory Bulb/surgery , Organ Culture Techniques , Phosphorylation/drug effects , Protein Kinase C/metabolism , Pyrrolidinones/therapeutic use , Receptor, Metabotropic Glutamate 5 , Receptors, Glutamate/metabolism , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/drug effects , Receptors, Metabotropic Glutamate/metabolism , Receptors, N-Methyl-D-Aspartate/agonists , Receptors, N-Methyl-D-Aspartate/metabolism
9.
Hippocampus ; 19(9): 844-54, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19253410

ABSTRACT

Galantamine, a novel Alzheimer's drug, is known to inhibit acetylcholinesterase activity and potentiate nicotinic acetylcholine receptor (nAChR) in the brain. We previously reported that galantamine potentiates the NMDA-induced currents in primary cultured rat cortical neurons. We now studied the effects of galantamine on long-term potentiation (LTP) in the rat hippocampal CA1 regions. The field excitatory postsynaptic potentials (fEPSPs) were induced by stimulation of the Schaffer collateral/commissural pathways in the hippocampal CA1 region. Treatment with 0.01-10 microM galantamine did not affect the slope of fEPSPs in the CA1 region. Galantamine treatment increased calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase Calpha (PKCalpha) activities with a bell-shaped dose-response curve peaked at 1 microM, thereby increasing the phosphorylation of AMPA receptor, myristoylated alanine-rich protein kinase C, and NMDA receptor as downstream substrates of CaMKII and/or PKCalpha. By contrast, galatamine treatment did not affect protein kinase A activity. Consistent with the bell-shaped CaMKII and PKCalpha activation, galantamine treatment enhanced LTP in the hippocampal CA1 regions with the same bell-shaped dose-response curve. Furthermore, LTP potentiation induced by galantamine treatment at 1 microM was closely associated with both CaMKII and PKC activation with concomitant increase in phosphorylation of their downstream substrates except for synapsin I. In addition, the enhancement of LTP by galantamine was accompanied with alpha7-type nAChR activation. These results suggest that galantamine potentiates NMDA receptor-dependent LTP through alpha7-type nAChR activation, by which the postsynaptic CaMKII and PKC are activated.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Galantamine/pharmacology , Long-Term Potentiation/drug effects , Nootropic Agents/pharmacology , Protein Kinase C-alpha/metabolism , Animals , Cyclic AMP-Dependent Protein Kinases/metabolism , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Excitatory Postsynaptic Potentials/drug effects , Galantamine/administration & dosage , Hippocampus/drug effects , Hippocampus/enzymology , Hippocampus/physiology , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Male , Membrane Proteins/metabolism , Myristoylated Alanine-Rich C Kinase Substrate , Neural Pathways/drug effects , Neural Pathways/enzymology , Neural Pathways/physiology , Nootropic Agents/administration & dosage , Phosphorylation/drug effects , Rats , Rats, Wistar , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, Nicotinic/metabolism , Synapsins/metabolism , alpha7 Nicotinic Acetylcholine Receptor
10.
Article in English | MEDLINE | ID: mdl-18941294

ABSTRACT

Tetrodotoxin (TTX), contained in puffer, has become an extremely popular chemical tool in the physiological and pharmacological laboratories since our discovery of its channel blocking action in the early 1960s. This brief review describes the history of discovery of TTX action on sodium channels, and represents a story primarily of my own work. TTX inhibits voltage-gated sodium channels in a highly potent and selective manner without effects on any other receptor and ion channel systems. TTX blocks the sodium channel only from outside of the nerve membrane, and is due to binding to the selectivity filter resulting in prevention of sodium ion flow. It does not impairs the channel gating mechanism. More recently, the TTX-resistant sodium channels have been discovered in the nervous system and received much attention because of their role in pain sensation. TTX is now known to be produced not by puffer but by bacteria, and reaches various species of animals via food chain.(Communicated by Masanori OTSUKA, M.J.A.).


Subject(s)
Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Tetrodotoxin/pharmacology , Action Potentials/drug effects , Animals , Ion Channel Gating , Tetraodontiformes
11.
Neurotoxicology ; 29(3): 546-55, 2008 May.
Article in English | MEDLINE | ID: mdl-18538410

ABSTRACT

The field of neurotoxicology needs to satisfy two opposing demands: the testing of a growing list of chemicals, and resource limitations and ethical concerns associated with testing using traditional mammalian species. National and international government agencies have defined a need to reduce, refine or replace mammalian species in toxicological testing with alternative testing methods and non-mammalian models. Toxicological assays using alternative animal models may relieve some of this pressure by allowing testing of more compounds while reducing expense and using fewer mammals. Recent advances in genetic technologies and the strong conservation between human and non-mammalian genomes allow for the dissection of the molecular pathways involved in neurotoxicological responses and neurological diseases using genetically tractable organisms. In this review, applications of four non-mammalian species, zebrafish, cockroach, Drosophila, and Caenorhabditis elegans, in the investigation of neurotoxicology and neurological diseases are presented.


Subject(s)
Animal Testing Alternatives , Nervous System Diseases/pathology , Toxicology/methods , Animals , Caenorhabditis elegans , Chloride Channels/drug effects , Environmental Pollutants/toxicity , Glutamic Acid/physiology , Insecta , Insecticides/toxicity , Manganese Poisoning/psychology , Pyrethrins/toxicity , Sodium Channels/drug effects , Zebrafish
12.
J Neurochem ; 106(3): 1092-103, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18445137

ABSTRACT

Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as cognitive-enhancing effect. We previously showed that nefiracetam potentiates NMDA-induced currents in cultured rat cortical neurons. To address questions whether nefiracetam affects NMDA receptor-dependent synaptic plasticity in the hippocampus, we assessed effects of nefiracetam on NMDA receptor-dependent long-term potentiation (LTP) by electrophysiology and LTP-induced phosphorylation of synaptic proteins by immunoblotting analysis. Nefiracetam treatment at 1-1000 nM increased the slope of fEPSPs in a dose-dependent manner. The enhancement was associated with increased phosphorylation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) without affecting synapsin I phosphorylation. In addition, nefiracetam treatment increased PKCalpha activity in a bell-shaped dose-response curve which peaked at 10 nM, thereby increasing phosphorylation of myristoylated alanine-rich protein kinase C substrate and NMDA receptor. Nefiracetam treatment did not affect protein kinase A activity. Consistent with the bell-shaped PKCalpha activation, nefiracetam treatment enhanced LTP in the rat hippocampal CA1 region with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with CaMKII and PKCalpha activation with concomitant increases in phosphorylation of their endogenous substrates except for synapsin I. These results suggest that nefiracetam potentiates AMPA receptor-mediated fEPSPs through CaMKII activation and enhances NMDA receptor-dependent LTP through potentiation of the post-synaptic CaMKII and protein kinase C activities. Together with potentiation of nicotinic acetylcholine receptor function, nefiracetam-enhanced AMPA and NMDA receptor functions likely contribute to improvement of cognitive function.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Hippocampus/enzymology , Long-Term Potentiation/physiology , Protein Kinase C/metabolism , Pyrrolidinones/pharmacology , Animals , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Hippocampus/drug effects , Long-Term Potentiation/drug effects , Male , Rats , Rats, Wistar
13.
Neurosci Lett ; 426(1): 54-8, 2007 Oct 09.
Article in English | MEDLINE | ID: mdl-17884292

ABSTRACT

Oseltamivir (Tamiflu) is now being stockpiled by several governments as a first line treatment for an anticipated outbreak of avian influenza caused by H5N1. However, abnormal behaviors and death associated with the use of Tamiflu have developed into a major issue in Japan where Tamiflu is often prescribed for seasonal influenza. Thus, it is critical to determine neuropsychiatric effects of oseltamivir and to establish methods for safe administration. Using juvenile rats and rat hippocampal slices, we investigated whether oseltamivir has adverse effects on the central nervous system. Systemic injection of oseltamivir (50mg/kg i.p.) produced no change in behavior within 2h. However, prior injection of oseltamivir significantly altered the duration of loss of lightning reflex following ethanol injection (3.3g/kg, i.p.). Ethanol injection in the presence of oseltamivir also resulted in enhanced hypothermia. In the CA1 region of hippocampal slices, oseltamivir (100 microM) induced paired-pulse facilitation in population spikes without changes in excitatory postsynaptic potentials. Similarly, 3 microM oseltamivir carboxylate, the active metabolite of oseltamivir, facilitated neuronal firing, though the facilitation did not involve GABAergic disinhibition. Moreover, oseltamivir carboxylate produced further facilitation following administration of 60mM ethanol. These findings indicate that oseltamivir has effects on the central nervous system, especially when combined with other agents.


Subject(s)
Brain/drug effects , Neurons/drug effects , Oseltamivir/pharmacology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Brain/metabolism , Brain/physiopathology , Central Nervous System Depressants/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Ethanol/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiopathology , Hypothermia/chemically induced , Hypothermia/metabolism , Hypothermia/physiopathology , Male , Neurons/metabolism , Organ Culture Techniques , Oseltamivir/metabolism , Pyramidal Cells/drug effects , Pyramidal Cells/metabolism , Rats , Reflex, Abnormal/drug effects , Reflex, Abnormal/physiology , Synaptic Transmission
14.
Alcohol Clin Exp Res ; 31(1): 89-99, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17207106

ABSTRACT

BACKGROUND: The gamma-aminobutyric acid-A (GABA(A)) receptor and glutamate receptors are among the most important target sites for the behavioral effects of ethanol. However, data in the literature concerning the ethanol modulation of the GABA(A) and glutamate receptors have been controversial. The activity of the neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) has recently been reported to be potently augmented by ethanol. The activation of nAChRs is also known to cause the release of various neurotransmitters including GABA and glutamate. Thus, ethanol potentiation of nAChRs is expected to stimulate the GABAergic and glutamatergic systems. METHODS: Whole-cell patch clamp experiments were performed using rat cortical neurons in primary culture to record spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs). RESULTS: Two types of neurons were distinguished: bipolar neurons possessed alpha4beta2 nAChRs generating a steady current in response to 30 nM ACh, and multipolar neurons that did not generate a current by ACh application. Acetylcholine greatly increased the frequency of mEPSCs and mIPSCs in bipolar neurons but not in multipolar neurons. The amplitude of neither type of neuron was affected by ACh. Ethanol at 10 to 100 mM suppressed the amplitude of mEPSCs while augmenting the amplitude of mIPSCs in both bipolar and multipolar neurons, indicating the direct action on the respective receptors. In bipolar neurons, ACh plus 100 mM ethanol greatly increased the frequency of mIPSCs beyond the levels achieved by ACh alone, while no such increases were observed in multipolar neurons. CONCLUSIONS: It is concluded that ethanol stimulation of nAChRs modulates the activity of both glutamate and GABA receptors in rat cortical bipolar neurons.


Subject(s)
Central Nervous System Depressants/pharmacology , Cerebral Cortex/physiology , Ethanol/pharmacology , Neurons/drug effects , Synaptic Transmission/drug effects , 2-Amino-5-phosphonovalerate/pharmacology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Cell Polarity/drug effects , Cerebral Cortex/cytology , Data Interpretation, Statistical , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Female , Patch-Clamp Techniques , Pregnancy , Rats , Rats, Sprague-Dawley
15.
Mol Pharmacol ; 71(2): 580-7, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17095583

ABSTRACT

Nicotinic acetylcholine receptors and N-methyl-D-aspartate (NMDA) receptors are known to be down-regulated in the brain of Alzheimer's disease patients. We have previously demonstrated that the nootropic drug nefiracetam potentiates the activity of both nicotinic acetylcholine and NMDA receptors and that nefiracetam modulates the glycine binding site of the NMDA receptor. Because the NMDA receptor is also modulated by Mg2+ and protein kinases, we studied their roles in nefiracetam action on the NMDA receptor by the whole-cell patch-clamp technique and immunoblotting analysis using rat cortical or hippocampal neurons in primary culture. The nefiracetam potentiation of NMDA currents was inhibited by the protein kinase C (PKC) inhibitor chelerythrine, but not by the protein kinase A (PKA) inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). In immunoblotting analysis, nefiracetam treatment increased the PKCalpha activity with a bell-shaped dose-response relationship peaking at 10 nM, thereby increasing phosphorylation of PKC substrate and NMDA receptor. Such an increase in PKCalpha-mediated phosphorylation was prevented by chelerythine. Nefiracetam treatment did not affect the PKA activity. Analysis of the current-voltage relationships revealed that nefiracetam at 10 nM largely eliminated voltage-dependent Mg2+ block and that this action of nefiracetam was sensitive to PKC inhibition. It was concluded that nefiracetam potentiated NMDA currents not by acting as a partial agonist but by interacting with PKC, allosterically enhancing glycine binding, and attenuating voltage-dependent Mg2+ block.


Subject(s)
Magnesium/pharmacology , Protein Kinase C/metabolism , Pyrrolidinones/pharmacology , Receptors, N-Methyl-D-Aspartate/agonists , Allosteric Regulation , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Electrochemistry , Glycine/metabolism , N-Methylaspartate , Neurons/cytology , Patch-Clamp Techniques , Phosphorylation/drug effects , Protein Kinase C/drug effects , Rats , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/physiology
16.
Neuropharmacology ; 51(7-8): 1181-91, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17011596

ABSTRACT

Several drugs are in clinical use for symptomatic treatment of Alzheimer's disease patients. Since Alzheimer's disease is known to be associated with down-regulation of the cholinergic and N-methyl-D-aspartate (NMDA) systems, most of these drugs inhibit acetylcholinesterase, potentiate the activity of nicotinic acetylcholine receptors (nAChRs), or modulate NMDA receptors. Galantamine is an anticholinesterase and allosterically potentiates the activity of the nicotinic receptors. We have recently found that galantamine potentiates the activity of NMDA receptors as well. Memantine is unique in that it inhibits the NMDA receptors. We have developed a hypothesis that combining galantamine and memantine will be more effective for improving the patient's conditions than monotherapy with either drug. Patch clamp and intracellular Ca(2+) imaging experiments using rat cortical and hippocampal neurons clearly provided the in vitro bases for our hypothesis. Memantine blocked the extrasynaptic NMDA receptor 100 times more potently than the synaptic NMDA receptor at negative membrane potentials and the block of both types of NMDA receptors was attenuated with depolarization. However, galantamine potentiation of the NMDA receptors was not voltage dependent. Thus, co-application of memantine with galantamine prevented the galantamine potentiation and the activation of extrasynaptic NMDA receptors, but membrane depolarization revealed the galantamine potentiation. Therefore, cell death is expected to be prevented by memantine near the resting potential while the NMDA-mediated synaptic transmission, which is down-regulated in the patients, is maintained and potentiated by galantamine. These results provide in vitro bases for the beneficial actions of galantamine and memantine combinations.


Subject(s)
Cholinesterase Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Galantamine/pharmacology , Memantine/pharmacology , Neuroprotective Agents/pharmacology , Nicotinic Agonists/pharmacology , Receptors, N-Methyl-D-Aspartate/physiology , 6-Cyano-7-nitroquinoxaline-2,3-dione/administration & dosage , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Action Potentials/drug effects , Alzheimer Disease/drug therapy , Animals , Bicuculline/pharmacology , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cells, Cultured/drug effects , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Cholinesterase Inhibitors/administration & dosage , Corpus Striatum/cytology , Corpus Striatum/embryology , Drug Evaluation, Preclinical , Drug Synergism , Excitatory Amino Acid Antagonists/administration & dosage , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Galantamine/administration & dosage , Glycine/pharmacology , In Vitro Techniques , Inhibitory Concentration 50 , Memantine/administration & dosage , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/physiology , Neuroprotective Agents/administration & dosage , Nicotinic Agonists/administration & dosage , Patch-Clamp Techniques , Perfusion/instrumentation , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/physiology , Strychnine/pharmacology , Synaptic Transmission/drug effects , Therapeutic Irrigation/instrumentation , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/antagonists & inhibitors
17.
J Pharmacol Exp Ther ; 319(1): 431-8, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16844844

ABSTRACT

The effects of ethanol on the GABA(A) receptors, which are regarded as one of the most important target sites of ethanol, are very controversial, ranging from potentiation to no effect. The delta subunit-containing GABA(A) receptors expressed in Xenopus oocytes were recently reported to be potently augmented by ethanol. We performed patch-clamp experiments using the cerebellar granule cells and mammalian cells expressing recombinant GABA(A) receptors. In granule cells, the sensitivity to GABA increased from 7 to 11 days in vitro. Furosemide, an antagonist of alpha6-containing GABA(A) receptors, inhibited GABA-induced currents more potently at 11 to 14 days than at 7 days. Ethanol at 30 mM had either no effect or an inhibitory effect on currents induced by low concentrations of GABA in granule cells. On alpha4beta2delta, alpha6beta2delta, or alpha6beta3deltaGABA(A) receptors expressed in Chinese hamster ovary cells, ethanol at 10, 30, and 100 mM had either no effect or an inhibitory effect on GABA currents. Ethanol inhibition of GABA(A) receptor was observed in all of the subunit combinations examined. In contrast, the perforated patch-clamp method to record the GABA currents revealed ethanol effects on the alpha6beta2delta subunits ranging from slight potentiation to slight inhibition. Ethanol seems to exert a dual action on the GABA(A) receptors and the potentiating action may depend on intracellular milieu. Thus, the differences between the GABA(A) receptors expressed in mammalian host cells and those in Xenopus oocytes in the response to ethanol might be due to changes in intracellular components under patch-clamp conditions.


Subject(s)
Cerebellum/drug effects , Ethanol/pharmacology , Receptors, GABA-A/drug effects , Animals , Azides/pharmacology , Benzodiazepines/pharmacology , CHO Cells , Cells, Cultured , Cricetinae , Furosemide/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/physiology , Recombinant Proteins/drug effects , Xenopus , gamma-Aminobutyric Acid/pharmacology
18.
Biophys Chem ; 120(1): 36-43, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16288955

ABSTRACT

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.


Subject(s)
Cell Membrane Permeability , Chloride Channels/chemistry , Chloride Channels/physiology , Chlorides/metabolism , Models, Biological , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Computer Simulation , Ion Transport , Models, Molecular , Sodium/pharmacokinetics , Sodium/physiology
19.
Hypertens Res ; 29(11): 923-34, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17345793

ABSTRACT

We examined the effects of angiotensin II (Ang II) on inward rectifier K+ currents (IK1) in rat atrial myocytes. [125I]Ang II-binding assays revealed the presence of both Ang II type 1 (AT1) and type 2 (AT2) receptors in atrial membrane preparations. Ang II inhibited IK1 in isolated atrial myocytes with an IC50 of 46 nmol/l. This inhibition was abolished by the AT, antagonist RNH6270 but not at all by the AT2 antagonist PD123319. Treatment of cells with pertussis toxin or a synthetic decapeptide corresponding to the carboxyl-terminus of Gialpha-3 abolished the inhibition by Ang II, indicating the role of a Gi-dependent signaling pathway. Accordingly, Ang II failed to inhibit IK1 in the presence of forskolin, dibutyryl-cAMP or protein kinase A catalytic subunits. In spite of the increased binding capacities for [125I]Ang II, Ang II failed to affect IKI in cells from spontaneously hypertensive rats (SHR). AT, immunoprecipitation from atrial extracts revealed decreased amounts of Gialpha-2 and Gialpha-3 proteins associated with this receptor in SHR as compared with controls. The reduced coupling of AT, with Gialpha. proteins may underlie the unresponsiveness of atrial IK1 to Ang II in SHR cells.


Subject(s)
Angiotensin II/pharmacology , Myocytes, Cardiac/drug effects , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Animals , Gene Expression , Heart Atria/cytology , In Vitro Techniques , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Receptor, Angiotensin, Type 1/metabolism , Signal Transduction/drug effects
20.
Hypertens Res ; 28(2): 173-9, 2005 Feb.
Article in English | MEDLINE | ID: mdl-16025745

ABSTRACT

Angiotensin II (Ang II) has been reported to indirectly influence atrial electrical activity and to play a critical role in atrial arrhythmias in hypertensive patients. However, it is unclear whether Ang II has direct effects on the electrophysiological activity of the atrium affected by hypertension. We examined the effects of Ang II on the action potentials of atrial myocytes enzymatically isolated from spontaneous hypertensive rats (SHRs). The action potentials were recorded by the perforated patch-clamp technique and the atrial expression of the receptors AT1a and AT2 was measured by radioimmunoassay. Ang II significantly shortened the action potential durations (APDs) of SHRs without changes in the resting membrane potentials (RMPs). Pretreatment with selective AT1a blockers abolished the Ang II-induced reduction of atrial APDs of SHRs; however, a selective AT2 blocker did not, which was consistent with the results of the receptor assay. Pretreatment with phosphatidylinositol 3 (PI3)-kinase inhibitor, phospholipase C inhibitor, or protein kinase C (PKC) inhibitor abolished the Ang II-induced shortening of atrial APDs, but pertussis toxin and protein kinase A (PKA) inhibitor did not. To study the effects of chronic AT1a inhibition on Ang II-induced shortening of atrial APD, SHRs were treated with AT1a blocker for 4 weeks. AT1a blocker abolished the Ang II-induced reduction of atrial APDs of SHRs and also significantly lowered their blood pressure. In conclusion, Ang II shortened atrial APDs of SHRs via AT1a coupled with the Gq-mediated inositol triphosphate (IP3)-PKC pathway. Our findings indicated that Ang II caused atrial arrhythmias in hypertensive patients by shortening the effective refractory period of the atrium.


Subject(s)
Action Potentials/drug effects , Angiotensin II/physiology , Hypertension/physiopathology , Myocytes, Cardiac/drug effects , Receptor, Angiotensin, Type 1/drug effects , Angiotensin II/pharmacology , Animals , Enzyme Inhibitors/pharmacology , Patch-Clamp Techniques , Radioimmunoassay , Rats , Rats, Inbred SHR , Receptor, Angiotensin, Type 2/drug effects
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