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.

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.

Forskolin Enhances Synaptic Transmission in Rat Dorsal Striatum through NMDA Receptors and PKA in Different Phases

The effect of forskolin on corticostriatal synaptic transmission was examined by recording excitatory postsynaptic currents (EPSCs) in rat brain slices using the whole-cell voltage-clamp technique. Forskolin produced a dose-dependent increase of corticostriatal EPSCs (1, 3, 10, and 30micrometer) immediately after its treatment, and the increase at 10 and 30micrometer was maintained even after its washout. When the brain slices were pre-treated with (DL)-2-amino-5-phosphonovaleric acid (AP-V, 100micrometer), an NMDA receptor antagonist, the acute effect of forskolin (10micrometer) was blocked. However, after washout of forskolin, an increase of corticostriatal EPSCs was still observed even in the presence of AP-V. When KT 5720 (5micrometer), a protein kinase A (PKA) inhibitor, was applied through the patch pipette, forskolin (10micrometer) increased corticostriatal EPSCs, but this increase was not maintained. When forskolin was applied together with AP-V and KT 5720, both the increase and maintenance of the corticostriatal EPSCs were blocked. These results suggest that forskolin activates both NMDA receptors and PKA, however, in a different manner.

Effects of Apigenin on Glutamate-induced Ca2+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 Ca2+concentration ([Ca2+]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 (1micrometerto 100micrometer for 5 min inhibited glutamate (100 micrometer 1 min) induced [Ca2+]i increase, concentration-dependently. Pretreatment with apigenin (30micrometer for 5 min significantly decreased the [Ca2+]i responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 micrometer 1 min) and N-methyl-D-aspartate (NMDA, 100 micrometer 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca2+]i response induced by 50 mM KCl solution, decreased the [Ca2+]i responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100micrometer 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca2+]i responses. Furthermore, treatment with apigenin (30micrometer significantly inhibited the amplitude and frequency of 0.1 mM [Mg2+o-induced [Ca2+]i spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia

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.

Fluoxetine Modulates Corticostriatal Synaptic Transmission through Postsynaptic Mechanism

Fluoxetine, widely used for the treatment of depression, is known to be a selective serotonin reuptake inhibitor (SSRI), however, there are also reports that fluoxetine has direct effects on several receptors. Employing whole-cell patch clamp techniques in rat brain slice, we studied the effects of fluoxetine on corticostriatal synaptic transmission by measuring the change in spontaneous excitatory postsynaptic currents (sEPSC). Acute treatment of rat brain slice with fluoxetine (10microM) significantly decreased the amplitude of sEPSC (84.1+/-3.3%, n=7), but did not alter its frequency (99.1+/-4.7%, n=7). Serotonin (10microM) also significantly decreased the amplitude (81.2+/-3.9%, n=4) of sEPSC, but did not affect its frequency (105.8+/-8.0, n=4). The effect of fluoxetine was found to have the same trend as that of serotonin. We also found that the inhibitory effect of fluoxetine on sEPSC amplitude (93.0+/-1.9%, n=8) was significantly blocked, but not serotonin (84.3+/-1.6%, n=4), when the brain slice was incubated with p-chloroamphetamine (10microM), which depletes serotonin from the axon terminals and blocks its reuptake. These results suggest that fluoxetine inhibits corticostriatal synaptic transmission through postsynaptic, and that these effects are exerted through both serotonin dependent and independent mechanism.

A Simple Method for Predicting Hippocampal Neurodegeneration in a Mouse Model of Transient Global Forebrain Ischemia

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.

NMDA Receptor-dependent Inhibition of Synaptic Transmission by Acute Ethanol Treatment in Rat Corticostriatal Slices

The effects of ethanol on corticostriatal synaptic transmission were examined, using extracellular recording and analysis of population spike amplitudes in rat brain slices, to study how acute ethanol intoxication impairs striatal function. Ethanol caused a decrease in population spike amplitudes in a dose dependent manner (50~200 mM). Pretreatment with picrotoxin, a gamma-amino butyric acid (GABA)A receptor antagonist, increased the population spikes but ethanol (100 mM) was still effective in decreasing the population spikes under this condition. In the presence of (DL)-2-amino-5-phosphonovaleric acid (APV), N-methyl-D-aspartate (NMDA) receptor antagonist, the inhibitory action of ethanol on population spikes was not shown. These results suggest that ethanol inhibits the glutamatergic corticostriatal synaptic transmission through blockade of NMDA receptors.

5-Hydroxytryptamine Inhibits Glutamatergic Synaptic Transmission in Rat Corticostriatal Brain Slice

Striatum is involved in the control of movement and habitual memory. It receives glutamatergic input from wide area of the cerebral cortex as well as an extensive serotonergic (5-hydroxytryptamine, 5-HT) input from the raphe nuclei. In our study, the effects of 5-HT on synaptic transmission were studied in the rat corticostriatal brain slice using in vitro whole-cell recording technique. 5-HT inhibited the amplitude as well as frequency of spontaneous excitatory postsynaptic currents (sEPSC) significantly, and neither gamma-aminobutyric acid (GABA) A receptor antagonist bicuculline (BIC), nor N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonovaleric acid (AP-V) could block the effect of 5-HT. In the presence non-NMDA receptor antagonist, 2, 3-dioxo-6-nitro-1, 2, 3, 4-tetrahydrobenxo[f] quinoxaline-7-sulfonamide (NBQX), the inhibitory effect of 5-HT was blocked. We also figured out that 5-HT change the channel kinetics of the sEPSC. There was a significant increase in the rise time during the 5-HT application. Our results suggest that 5-HT has an effect on both pre- and postsynaptic site with decreasing neurotransmitter release probability of glutamate and decreasing the sensitivity to glutamate by increasing the rise time of non-NMDA receptor mediated synaptic transmission in the corticostriatal synapses.

Dopamine Modulates Corticostriatal Synaptic Transmission through Both D1 and D2 Receptor Subtypes in Rat Brain

Striatum has important roles in motor control, habitual learning and memory. It receives glutamatergic inputs from neocortex and thalamus, and dopaminergic inputs from substantia nigra. We examined effects of dopamine (DA) on the corticostriatal synaptic transmission using in vitro extracellular recording technique in rat brain corticostriatal slices. Synaptic responses were elicited by stimulation of cortical glutamatergic inputs on the corpus callosum and recorded in the dorsal striatum. Corticostriatal population spike (PS) amplitudes were decreased (39.4+/-7.9%) by the application of 100microM DA. We applied receptor subtype specific agonists and antagonists and characterized the modulation of corticostriatal synaptic transmission by different DA receptor subtypes. D2 receptor agonist (quinpirole), antagonist (sulpiride), and D1 receptor antagonist (SKF 83566), but not D1 receptor agonist (SKF 38393), induced significantly the reduction of striatal PS. Pretreatment neither with SKF 83566 nor sulpiride significantly affected corticostriatal synaptic inhibition by DA. However, the inhibition of DA was completely blocked by pretreatment with mixed solution of both SKF 83566 and sulpiride. These results suggest that DA inhibits corticostriatal synaptic transmission through both D1 and D2 receptors in concert with each other.

Inhibitory Modulation of 5-Hydroxytryptamine on Corticostriatal Synaptic Transmission in Rat Brain Slice

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.

Characterization of Norepinephrine Release in Rat Posterior Hypothalamus Using in vivo Brain Microdialysis

In the present study, we used the microdialysis technique combined with high performance liquid chromatography (HPLC) and electrochemical detection to measure the extracellular levels of norepinephrine (NE) in the posterior hypothalamus in vivo, and to examine the effects of various drugs, affecting central noradrenergic transmission, on the extracellular concentration of NE in the posterior hypothalamus. Microdialysis probes were implanted stereotaxically into the posterior hypothalamus (coordinates: posterior 4.3 mm, lateral 0.5 mm, ventral 8 mm, relative to bregma and the brain surface, respectively) of rats, and dialysate collection began 2 hr after the implantation. The baseline level of monoamines in the dialysates were determined to be: NE 0.17 +/- 0.01, 3,4-dihydroxyphenylacetic acid (DOPAC) 0.94 +/- 0.07, homovanillic acid (HVA) 0.57 +/- 0.05 pmol/sample (n=8). When the posterior hypothalamus was perfused with 90 mM potassium, maximum 555% increase of NE output was observed. Concomitantly, this treatment significantly decreased the output of DOPAC and HVA by 35% and 28%, respectively. Local application of imipramine (50microM) enhanced the level of NE in the posterior hypothalamus (maximum 200%) compared to preperfusion control values. But, DOPAC and HVA outputs remained unchanged. Pargyline, an irreversible monoamine oxidase inhibitor, i.p. administered at a dose of 75 mg/kg, increased NE output (maximum 165%), while decreased DOPAC and HVA outputs (maximum 13 and 12%, respectively). These results indicate that NE in dialysate from the rat posterior hypothalamus were neuronal origin, and that manipulations which profoundly affected the levels of extracellular neurotransmitter had also effects on metabolite levels.

Apoptosis Induced by Chemotherapeutic Agents in Differentiated HL-60 Human Leukemic Cell Line / 대한소아혈액종양학회지

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.

Primary role of posterior hypothalamic cholinergic receptors in central regulation of blood pressure and heart rate in rats

The purpose of the present study is to determine the role of muscarinic cholinergic receptors of posterior hypothalamus in the central blood pressure regulation when respiration is controlled. In anesthetized and artificially ventilated rats, vasodepressor response was evoked by injection of L-glutamate (10 nmol) neuroexcitatory amino acid into the posterior hypothalamic area. The injection of carbachol (0.5 ~ 8 nmol) into the same area induced dose-dependent vasodepressor and bradycardic responses. Pretreatment with atropine (4 nmol) completely blocked the vasodepressor response to carbachol (2 nmol). In contrast, in spontaneously breathing rats, the injection of carbachol (8 nmol) into the posterior hypothalamic area induced the vasopressor and tachycardic responses. These results suyggest that the muscarinic cholinergic receptors in the posterior hypothalamic area primarily play an inhibitory role in the central regulation of blood pressure and heart rate.

Apoptotic process is involved in the L-glutamate-induced PC12 cell death

Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying excitatory amino acid-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which L-glutamate, an excitatory amino acid neurotransmitter, induces cell death in PC12 cell lines. To characterize cell death, we employed sandwich enzyme-linked immunosorbent assay (ELISA) method for cellular DNA fragmentation, DNA agarose gel electrophoresis and chromatin staining by acridine orange and ethidium bromide after treating the PC12 cells with L-glutamate. L-Glutamate caused dose-dependent cell death with a maximum at 24 hrs after the treatment. These cellular fragmentation was blocked by pretreatment of MK-801, a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, and nerve growth factor(NGF). Analysis of DNA integrity from L-glutamate-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. The PC12 cells that were induced to die by L-glutamate treatment exhibited classical chromatin condensation under the light microscopy after acridine orange and ethidium bromide staining. These results suggest that apoptosis is one of the key features that are involved in L-glutamate-induced excitotoxic cell death in PC12 cells, and these cell death are mediated by NMDA receptor and depend on NGF.

Enhanced coupling of M-1 muscarinic receptors to activation of phospholipase C upon mutation of a transposed amino acid triplet repeat

The C-terminus ends of the second putative transmembrane domains of both M-1 and M-2 Muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in M-1 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-LYT) in the sequence Of M-2 receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of M-1 receptors to the corresponding M-2 receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT M-1 receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant M-1 receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant M-1 receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in M-1 muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of M-1 receptors to the stimulation of phospholipase C.