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.
Gastric Emptying ; Methods ; Serotonin

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.
Antidepressive Agents ; Anxiety Disorders ; Citalopram ; Depression ; Methods ; Neuroblastoma ; Serotonin

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.
Dopamine ; Epilepsy ; Neuroblastoma* ; Receptors, Serotonin, 5-HT3 ; Serotonin

No abstract available.
Chromatography, Liquid* ; Ciprofloxacin* ; Therapeutic Equivalency*

Striatum plays a crucial role in the movement control and habitual learning. It receives an information from wide area of cerebral cortex as well as an extensive serotonergic (5-hydroxytryptamine, 5-HT) input from raphe nuclei. In the present study, the effects of 5-HT to modulate synaptic transmission were studied in the rat corticostriatal brain slice using in vitro extracellular recording technique. Synaptic responses were evoked by stimulation of cortical glutamatergic inputs on the corpus callosum and recorded in the dorsal striatum. 5-HT reversibly inhibited coticostriatal glutamatergic synaptic transmission in a dose-dependent fashion (5, 10, 50, and 100 microM), maximally reducing in the corticostriatal population spike (PS) amplitude to 40.1+/-5.0% at a concentration of 50 microM 5-HT. PSs mediated by non-NMDA glutamate receptors, which were isolated by bath application of the NMDA receptor antagonist, d, l-2-amino-5-phospohonovaleric acid (AP-V), were decreased by application of 50 microM 5-HT. However, PSs mediated by NMDA receptors, that were activated by application of zero Mg2+ aCSF, were not significantly affected by 50 microM 5-HT. To test whether the corticostriatal synaptic inhibitions by 5-HT might involve a change in the probability of neurotransmitter release from presynaptic nerve terminals, we measured the paired-pulse ratio (PPR) evoked by 2 identical pulses (50 ms interpulse interval), and found that PPR was increased (33.4+/-5.2%) by 5-HT, reflecting decreased neurotransmitter releasing probability. These results suggest that 5-HT may decrease neurotransmitter release probability of glutamatergic corticostriatal synapse and may be able to selectively decrease non-NMDA glutamate receptor-mediated synaptic transmission.
Animals ; Baths ; Brain* ; Cerebral Cortex ; Corpus Callosum ; Depression ; Glutamic Acid ; Learning ; N-Methylaspartate ; Neurotransmitter Agents ; Raphe Nuclei ; Rats* ; Receptors, Glutamate ; Receptors, N-Methyl-D-Aspartate ; Serotonin* ; Synapses ; Synaptic Transmission*

In the present study, we developed a simple method to predict the neuronal cell death in the mouse hippocampus and striatum following transient global forebrain ischemia by evaluating both cerebral blood flow and the plasticity of the posterior communicating artery (PcomA). Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery (BCCAO) for 30 min. The regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry. The plasticity of PcomA was visualized by intravascular perfusion of India ink solution. When animals had the residual cortical microperfusion less than 15% as well as the smaller PcomA whose diameter was less than one third compared with that of basilar artery, neuronal damage in the hippocampal subfields including CA1, CA2, and CA4, and in the striatum was consistently observed. Especially, when mice met these two criteria, marked neuronal damage was observed in CA2 subfield of the hippocampus. In contrast, after transient BCCAO, neuronal damage was consistently produced in the striatum, dependent more on the degree of rCBF reduction than on the plasticity of PcomA. The present study provided simple and highly reproducible criteria to induce the neuronal cell death in the vulnerable mice brain areas including the hippocampus and striatum after transient global forebrain ischemia.
Animals ; Arteries ; Basilar Artery ; Brain ; Carotid Artery, Common ; Cell Death ; Halothane ; Hippocampus ; Humans ; India ; Ink ; Ischemia* ; Laser-Doppler Flowmetry ; Male ; Mice* ; Neurons ; Perfusion ; Plastics ; Prosencephalon*

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.
Acepromazine* ; Arrhythmias, Cardiac ; Humans* ; Kidney* ; Long QT Syndrome ; Patch-Clamp Techniques ; Potassium Channels* ; Potassium* ; Tail

No abstract available.
External Fixators

Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion.
Animals ; Antioxidants/*metabolism ; Catalase/metabolism ; Enzyme Activation ; Glutathione Peroxidase/metabolism ; Hippocampus/*blood supply ; Hydrogen Peroxide/metabolism ; Hydroxybenzoic Acids/metabolism ; Hydroxyl Radical/*metabolism ; Ischemic Attack, Transient/*metabolism/physiopathology/prevention & control ; *Ischemic Preconditioning ; Male ; *Prosencephalon ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism/prevention & control

PURPOSE: Chemotherapeutic agents are known to induce cell death in cancer cells by apoptotic mechanisms. This study was to investigate the influence of the differentiation on the apoptotic potential of chemotherapeutic agents. METHODS: Etoposide and cytosine arabinoside (Ara-C) were chosen as chemotherapeutic agents, and human promyelocytic leukemia cell line, HL-60, was used as target cells. RESULTS: Etoposide or Ara-C treated HL-60 cells showed cytoplasmic blebbing and nuclear condensation and fragmentation under fluorescence microscope when stained with acridine orange/ethidium bromide. In addition, the cellular DNA of HL-60 cells was found to cleave into internucleosomal fragments after treatment with chemotherapeutic agents. These findings were the characteristics of apoptosis and suggested the induction of apoptotic cell death of HL-60 cells by etoposide or Ara-C treatment. HL-60 cells are known to differentiate into myeloid or monocytic lineage by retinoic acid, phorbol 12-myristate acetate (PMA) and dimethyl sulfoxide (DMSO), and this differentiation itself can activate apoptosis program, so-called 'apoptosis by terminal differentiation'. The effect of terminal differentiation by PMA or DMSO on the apoptosis induced by etoposide or Ara-C was also investigated, utilizing qualitative and quantitative DNA fragmentation assay. HL-60 cells treated with PMA (100 nM) were adherent to culture dish and formed cellular processes. DMSO (1.25%) treated HL-60 cells instead recovered the ability to reduce nitroblue tetrazolium to blue-to-purple formazan, indicating its differentiation. After induction of differentiation by PMA or DMSO, differentiated HL-60 cells were treated with etoposide (10 muM) and Ara-C (50 muM) to compare its apoptotic potential with that of undifferentiated HL-60 cells. The ladder DNA induced by etoposide and Ara-C was decreased in differentiated HL-60 cells. On quantitative analysis of DNA fragmentation, PMA reduced DNA fragmentation induced by etoposide and Ara-C to 73% and 69%, respectively, and DMSO reduced it to 74% and 56%, respectively. In western blot analysis, the expression of Bcl-2, which is known to inhibit etoposide and Ara-C induced apoptosis, decreased significantly in HL-60 cells differentiated by PMA or DMSO. CONCLUSION: These results suggest that the differentiation of HL-60 cells by PMA or DMSO prevents apoptosis by etoposide and Ara-C, but bcl-2 proto-oncogene may have only minor role in inhibiting apoptosis by chemotherapeutic agents in differentiated HL-60 cell.
Apoptosis* ; Blister ; Blotting, Western ; Cell Death ; Cell Line* ; Cytarabine ; Cytoplasm ; Dimethyl Sulfoxide ; DNA ; DNA Fragmentation ; Etoposide ; Fluorescence ; HL-60 Cells ; Humans* ; Leukemia ; Nitroblue Tetrazolium ; Proto-Oncogenes ; Tretinoin