Anti-Inflammatory Effects of Miyako Bidens pilosa in a Mouse Model of Amyotrophic Lateral Sclerosis and Lipopolysaccharide-Stimulated BV-2 Microglia.

Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-ß and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

Functional Modulation of Nav1.2 Voltage-Gated Sodium Channels Induced by Escitalopram.

Escitalopram, a selective serotonin reuptake inhibitor (SSRI), may induce seizures, particularly in epileptic patients. In this study, we investigated the effect of escitalopram in Nav1.2 voltage-gated sodium channels (VGSCs) transfected HEK293 cells. Nav1.2 VGSCs current decreased by approximately 50.7±8.3% under treatment with 100 µM escitalopram. The IC50 of escitalopram against Nav1.2 VGSCs was 114.17 µM. Moreover, the treatment with 100 µM escitalopram changed the voltage-dependence of inactivation and the voltage at half-maximal inactivation shifted significantly from -50.3±3.7 to -56.7±6.0 mV toward negative potential under treatment with 100 µM escitalopram. Surprisingly, the treatment with 100 µM escitalopram also changed the voltage-dependence of activation and the voltage at half-maximal activation shifted significantly from -13.8±4.6 to -21.5±3.9 mV toward negative potential under treatment with 100 µM escitalopram. These findings suggested that escitalopram might be able to inhibit Nav1.2 VGSCs current and affects both activation and inactivation states of Nav1.2 VGSCs.

Corticosterone Inhibits the Proliferation of C6 Glioma Cells via the Translocation of Unphosphorylated Glucocorticoid Receptor.

Astroglial cells have been considered to have passive brain function by helping to maintain neurons. However, recent studies have revealed that the dysfunction of such passive functions may be associated with various neuropathological diseases, such as schizophrenia, Alzheimer's disease, amyotrophic lateral sclerosis and major depression. Corticosterone (CORT), which is often referred to as the stress hormone, is a well-known regulator of peripheral immune responses and also shows anti-inflammatory properties in the brain. However, it is still obscure how CORT affects astroglial cell function. In this study, we investigated the effects of CORT on the proliferation and survival of astroglial cells using C6 glioma cells. Under treatment with CORT for 24h, the proliferation of C6 glioma cells decreased in a dose-dependent manner. Moreover, this inhibition was diminised by treatment with mifepristone, a glucocorticoid receptor (GR) antagonist, but not by spironolactone, a mineralocorticoid receptor (MR) antagonist, and was independent of GR phosphorylation and other GR-related intracellular signaling cascades. Furthermore, it was observed that the translocation of GR from the cytosol to the nucleus was promoted by the treatment with CORT. These results indicate that CORT decreases the proliferation of C6 glioma cells by modifying the transcription of a particular gene related to cell proliferation independent of GR phosphorylation.

Effect of lamotrigine on Na(v)1.4 voltage-gated sodium channels.

Lamotrigine (LTG) is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder and it has been known that LTG targets voltage-dependent sodium channels (VGSCs). In this study, we investigated the effect of LTG on the Nav1.4 Na(+) current using HEK293 cells expressing mouse Nav1.4 VGSCs. By the treatment of LTG, Nav1.4 Na(+) current was inhibited in a dose-dependent manner. Moreover, 100 µM LTG decreased Nav1.4 Na(+) current around 40% and shifted the V1/2 of the inactivation curve to the hyperpolarization side by 20.96 mV. These findings suggest that LTG inhibits Nav1.4 Na(+) current and modifies the kinetics of the inactivated state.

Corticosterone suppresses the proliferation of RAW264.7 macrophage cells via glucocorticoid, but not mineralocorticoid, receptor.

Macrophages are white blood cells within tissues that are produced by monocytes and help to protect against infection by bacteria through phagocytosis. Several studies have shown a correlation between the state of depression and abnormalities in the immune response. Corticosterone (CORT), which is often referred to as the stress hormone, is a well-known regulator of peripheral immune responses and also shows anti-inflammatory properties in the body. However, it is still unclear how CORT regulates macrophage function. In this study, we focused on the effects of CORT on the proliferation and survival of macrophage cells using the macrophage cell line RAW264.7. Under treatment with 10 µM CORT for 24 h, the proliferation of RAW264.7 cells decreased to 73.6% of that in the control. Moreover, this inhibition was blocked by treatment with mifepristone, a glucocorticoid receptor (GR) antagonist, but not by spironolactone, a mineralocorticoid receptor (MR) antagonist. In an lactate dehydrogenase (LDH) assay, CORT did not show any cytotoxic effect on RAW264.7 cells. JC-1 cell staining also showed that CORT did not influence mitochondrial dysfunction in RAW264.7 cells. In an investigation of the modulation of a signaling cascade by CORT, treatment with CORT promoted the translocation of GR, but not MR, from the cytosol to the nucleus in RAW264.7 cells. In conclusion, our findings suggest that CORT suppresses the proliferation of RAW264.7 cells by controlling the transcription of a particular gene, which is related to cell proliferation, through the formation of a CORT-GR complex.

Inhibitory effect of atomoxetine on Nav1.2 voltage-gated sodium channel currents.

BACKGROUND: Atomoxetine (ATX), a norepinephrine reuptake inhibitor (NRI), is used to attenuate the symptoms of Attention Deficit/Hyperactivity Disorder (AD/HD) by increasing neurotransmitter concentrations at the synaptic cleft. Although Nav1.2 voltage-gated sodium channels (VGSCs) are thought to play a role in monoamine transmitter release in the synaptic junction, it is unclear how atomoxetine affects Nav1.2 VGSCs. METHODS: In this study, we investigated the effect of ATX on Nav1.2 VGSC-transfected HEK293 cells with the whole-patch clamp technique. RESULTS: Nav1.2 VGSC current decreased by 51.15 ± 12.75% under treatment with 50 µM ATX in the resting state (holding membrane potential at - 80 mV). The IC50 of ATX against Nav1.2 VGSC current was 45.57 µM. The activation/inactivation curve of Nav1.2 VGSC currents was shifted toward hyperpolarization by 50 µM ATX. In addition, the inhibitory effect of ATX increased with membrane depolarization (holding membrane potential at - 50 mV) and its IC50 was 10.16 µM. Moreover, ATX showed the time-dependent interaction in the inactivation state. CONCLUSION: These findings suggest that ATX interacts with Nav1.2 VGSCs producing the inhibition of current and the modification of kinetic properties in the state-dependent manner.

[The lipid metabolism abnormality in patients administered with olanzapine].

The atypical antipsychotic medication olanzapine is a useful agent in acute and maintenance treatment of schizophrenia and related disorders. It has beneficial effects on both positive and negative symptoms, an early onset of antipsychotic action and a favourable side effect profile. On the other hand, olanzapine has many reports of causing weight gain, glucose metabolism disturbances and lipidosis. We carried out blood tests (leptin, adiponectin, remnant-like lipoprotein cholesterol (RLP-C), total cholesterol, HbA1C, 75-OGTT and etc.) on patients with schizophrenia who had taken olanzapine. As a result, leptin, neutral lipid and RLP-C were significantly correlated by BMI. (The average blood test data and BMI revealed a normal range). Most analysis results of the lipoprotein fraction by a polyacrylamide-gel-electrophoresis method were normal patterns. Furthermore, the serum insulin concentrations from 75 g glucose tolerance (75 g-OGTT) 30 minutes later, in one third of patients receiving olanzapine, registered more than 100 microU/ml. The mechanism of the insulin secretion rise by olannzapine is unknown. Olanzapine may impair glucose tolerance due in part to increased insulin resistance. These findings do not necessarily imply that olanzapine is directly associated with a risk of impairment of weight gain, glucose metabolism disturbances and lipidosis. These results suggest that it is useful to promote diet cure and exercise therapy with patients with high BMI levels.

Duloxetine ameliorates lipopolysaccharide-induced microglial activation by suppressing iNOS expression in BV-2 microglial cells.

RATIONALE: It is known that both selective serotonin and serotonin noradrenaline reuptake inhibitors (SSRI, SNRI) are first-line drugs for the treatment of major depressive disorder. It has also been considered that both SSRI and SNRI can improve the symptoms of major depressive disorder by increasing the concentration of monoamine in the synaptic cleft based on the monoamine hypothesis. However, accumulating evidence has indicated that inflammation in the brain may be a key factor in the pathophysiological mechanisms that underlie the development of major depressive disorder. OBJECTIVES: It has been advocated that microglial cells may regulate the inflammatory response under pathological conditions such as major depressive disorder. In this study, we focused on whether duloxetine can ameliorate the inflammatory response induced by lipopolysaccharide (LPS) in BV-2 microglial cells. RESULTS: Our results indicated that duloxetine significantly decreased the NO production induced by LPS. The increase in the protein expression level of iNOS induced by LPS was significantly decreased by treatment with duloxetine. Moreover, the increases in the protein expression levels of phosphorylated-IκBα, phosphorylated-Akt and Akt induced by LPS were also significantly decreased. Unexpectedly, the protein expression levels of other pro-inflammatory factors such as COX-2 and the phosphorylation ratios for various molecules including IκBα and Akt were not changed by treatment with duloxetine. CONCLUSIONS: These findings suggest that duloxetine may have an anti-inflammatory effect, which could contribute to its therapeutic effectiveness for major depressive disorder.

Contributions of S- and R-citalopram to the citalopram-induced modulation of the function of Nav1.5 voltage-gated sodium channels.

Citalopram, a selective serotonin reuptake inhibitor (SSRI), has been reported to have adverse effects such as cardiotoxicity, including prolongation of the QTc interval. Although citalopram is well known to be a racemic compound comprised of S-citalopram (escitalopram) and R-citalopram, it is still unclear which enantiomer is responsible for cardiotoxicity induced by citalopram. It is also unclear which biomolecule is the target that produces the adverse effect of citalopram. In this study, we investigated whether citalopram, escitalopram and R-citalopram had an electrophysiological effect on Nav1.5 voltage-gated sodium channel (VGSC) current and how their electrophysiological properties affected Nav1.5 VGSC. To examine the effects of the electrophysiological properties of them, whole-cell patch clamp recording was performed using HEK293 cells expressing human Nav1.5 VGSCs. Nav1.5 VGSC current decreased by 60.0 ± 6.3% and 55.1 ± 12.5% under treatment with 100 µM citalopram and escitalopram, respectively. However, 100 µM R-citalopram decreased Nav1.5 VGSC current by only 36.2 ± 8.7%. In addition, treatment with 100 µM citalopram and escitalopram changed the voltage-dependence of activation and induced a negative shift of the voltage of half-maximal activation compared to 100 µM R-citalopram. In contrast, treatment with 100 µM citalopram and escitalopram, but not R-citalopram, changed the voltage-dependence of inactivation, and the voltage at half-maximal inactivation slightly shifted toward negative potential. These results suggest that the adverse cardiac effect produced by citalopram might result from modification of the electrophysiological properties of Nav1.5 VGSCs, and escitalopram might contribute more to this adverse effect than R-citalopram.

Neoline, an active ingredient of the processed aconite root in Goshajinkigan formulation, targets Nav1.7 to ameliorate mechanical hyperalgesia in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Goshajinkigan (GJG), a traditional Japanese Kampo formula, has been shown to exhibit several pharmacological actions, including antinociceptive effects. Processed aconite root (PA), which is considered to be an active ingredient of GJG, has also been demonstrated to have an ameliorative effect on pain, such as diabetic peripheral neuropathic pain. We recently identified neoline as the active ingredient of both GJG and PA that is responsible for its effects against oxaliplatin-induced neuropathic pain in mice. AIM OF THE STUDY: In the present study, we investigated whether GJG, PA, and neoline could inhibit Nav1.7 voltage-gated sodium channel (VGSC) current and whether neoline could ameliorate mechanical hyperalgesia in diabetic mice. MATERIALS AND METHODS: To assess the electrophysiological properties of GJG extract formulation, powdered PA, and neoline on Nav1.7 VGSCs, whole-cell patch clamp recording was performed using human HEK293 cells expressing Nav1.7 VGSCs. In addition, the ameliorative effects of neoline on diabetic peripheral neuropathic pain were evaluated using the von Frey test in streptozotocin (STZ)-induced diabetic model mice. RESULTS: GJG extract formulation significantly inhibited Nav1.7 VGSC peak current. Powdered PA also inhibited Nav1.7 VGSC peak current. Like GJG and PA, neoline could inhibit Nav1.7 VGSC current. When diabetic mice were treated with neoline by intraperitoneal acute administration, the mechanical threshold was increased in diabetic mice, but not in non-diabetic mice, in a behavioral study. CONCLUSION: These results suggest that neoline might be a novel active ingredient of GJG and PA that is one of responsible ingredients for ameliorating mechanical hyperalgesia in diabetes via the inhibition of Nav1.7 VGSC current at least.

Mutant gammaPKC found in spinocerebellar ataxia type 14 induces aggregate-independent maldevelopment of dendrites in primary cultured Purkinje cells.

Missense mutations in protein kinase Cgamma (gammaPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant gammaPKC found in SCA14 is susceptible to aggregation and induces apoptosis in cultured cell lines. In the present study, we investigated whether mutant gammaPKC formed aggregates and how mutant gammaPKC affects the morphology and survival of cerebellar Purkinje cells (PCs), which are degenerated in SCA14 patients. Adenovirus-transfected primary cultured PCs expressing mutant gammaPKC-GFP also had aggregates and underwent apoptosis. Long-term time-lapse observation revealed that PCs have a potential to eliminate aggregates of mutant gammaPKC-GFP. Mutant gammaPKC-GFP disturbed the development of PC dendrites and reduced synapse formation, regardless of the presence or absence of its aggregates. In PCs without aggregates, mutant gammaPKC-GFP formed soluble oligomers, resulting in reduced mobility and attenuated translocation of mutant gammaPKC-GFP upon stimulation. These molecular properties of mutant gammaPKC might affect the dendritic morphology in PCs, and be involved in the pathogenesis of SCA14.

Cell-dependent physiological synaptic action of morphine in the rat habenular nucleus: morphine both inhibits and facilitates excitatory synaptic transmission.

Although several lines of evidence have suggested that the activity of thalamic neurons is modulated by opioids, the mechanism by which morphine in the thalamus regulates the release of excitatory neurotransmitters remains unclear. In the present study, we investigated the synaptic modulation of morphine to regulate excitatory synaptic transmission, probably glutamatergic transmission, in habenular nucleus (Hb) and centrolateral nucleus (CL) neurons in the rat thalamus. Using the whole-cell patch-clamp technique, we found dual modulation by morphine in Hb neurons: morphine caused either inhibition or facilitation of the miniature excitatory postsynaptic current (mEPSC) frequency in the Hb. In Hb neurons that showed a morphine-induced decrease in the mEPSC frequency, the mEPSC amplitude was also decreased in the presence of morphine. In contrast, the mEPSC amplitude was markedly increased in Hb neurons that showed a morphine-induced increase in the mEPSC frequency. We also observed a significant decrease in the mEPSC frequency with morphine in CL neurons without any change in the mEPSC amplitude, whereas morphine did not facilitate the mEPSC frequency in CL neurons. These results suggest that morphine may induce cell-dependent dual modulation of glutamatergic synaptic transmission in the Hb.

mu-Opioid receptor-independent fashion of the suppression of sodium currents by mu-opioid analgesics in thalamic neurons.

Most reports in the literature have shown that the effects of opioid analgesics are primarily mediated by mu-opioid receptor (MOR), whereas other potential targets of opioid analgesics have not been thoroughly characterized. In this study, we found that extracellular application of morphine, fentanyl or oxycodone, which are all considered to be MOR agonists, at relatively high concentrations, but not endogenous mu-opioid peptides, produced a concentration-dependent suppression of sodium currents in cultured thalamic neurons. These effects of opioids were not affected by either a MOR antagonist naloxone or a deletion of MOR gene. Among these opioids, fentanyl strongly suppressed sodium currents to the same degree as lidocaine, and both morphine and oxycodone slightly but significantly reduced sodium currents when they were present extracellularly. In contrast, the intracellular application of morphine, but not oxycodone, fentanyl or lidocaine, reduced sodium currents. These results suggest that morphine, fentanyl and oxycodone each produce the MOR-independent suppression of sodium currents by distinct mechanisms in thalamic neurons.

The C-terminal region of serotonin transporter is important for its trafficking and glycosylation.

We investigated the effects of brefeldin A and ilimaquinone, inhibitors of membrane trafficking, using serotonin transporter (SERT)-expressing COS-7 cells. Both drugs significantly inhibited the serotonin uptake activity of SERT and caused SERT to be retained in the endoplasmic reticulum (ER), indicating that membrane trafficking is an important factor for SERT functional regulation. In agreement with previous reports, a C-terminal-deletion mutant of SERT (SERTDeltaCT) mostly localized to the ER and completely lacked serotonin uptake activity. To further elucidate the role of the C-terminus of SERT, we investigated whether overexpression of FLAG-tagged SERT C-terminus (FLAG-SERT-CT) affected the serotonin uptake activity and glycosylation of SERT. Interestingly, when concomitantly expressed with full-length FLAG-SERT in COS-7 cells, FLAG-SERT-CT increased the serotonin uptake activity and mature glycosylation of FLAG-SERT. These results indicate that the C-terminal region of SERT plays a crucial role in the functional regulation of SERT via membrane trafficking and glycosylation. In addition, proteasome inhibitors induced apparent ER stress, significantly decreased the serotonin uptake activity and mature glycosylation of SERT and caused SERT to be localized to the ER, suggesting that SERT function would be attenuated via membrane trafficking in pathological states that trigger ER stress.

Post-synaptic action of morphine on glutamatergic neuronal transmission related to the descending antinociceptive pathway in the rat thalamus.

Morphine is a prototypical mu-opioid receptor (MOR) agonist, and can directly inhibit pain transmission at both spinal and supraspinal levels. In the present study, we investigated the properties of thalamic neurons in an opioid-sensitive pain-modulating circuit. Application of morphine to cultured thalamic neurons evoked a potentiation of glutamate-induced peak currents, which was blocked by the MOR antagonist. Application of the protein kinase C inhibitor chelerythrine significantly inhibited the morphine-evoked enhancement of glutamate-induced currents. Immunoreactivity for MOR was observed with high density in the habenular nucleus (Hb) of the thalamus in rats, which was clearly co-localized with NMDA receptor subunit 1 (NRI). In this study, we show that microinjection of morphine into the Hb produced a dose-dependent increase in the tail-flick latency and enhanced the antinociceptive effect induced by the intra-Hb injection of glutamate. When fluoro-gold (FG) was used as a retrograde tracer, we found that FG-labeled neurons in the Hb after the microinjection of FG into the periaqueductal gray expressed both MOR and NR1. The present data suggest that the stimulation of MOR in the Hb may be involved in activation of the descending antinociceptive pathway through glutamatergic neurotransmission via the NMDA receptor.

Chronic pain-induced astrocyte activation in the cingulate cortex with no change in neural or glial differentiation from neural stem cells in mice.

Pain pathways terminate in discrete brain areas that monitor the sensory and affective qualities of the initiating stimulus and show remarkable plasticity. Here, we found that chronic pain by sciatic nerve ligation caused a dramatic increase in glial fibrillary acidic protein (GFAP)-like immunoreactivity (IR), which is located in the dendritic astrocytes, with its expanding distribution in the cingulate cortex (CG) of mice. The branched GFAP-like IR in the CG of nerve-ligated mice was overlapped with S100beta-like IR, which is highly limited to the cell body of astrocytes, whereas there was no difference of S100beta-like IR between sham-operated and nerve-ligated mice. The number of BrdU-positive cells on the CG was not changed by sciatic nerve ligation. Furthermore, subventricular zone (SVZ)-derived neural stem cells marked by pEGFP-C1 did not migrate toward the CG after sciatic nerve ligation. In the behavioral assay, the thermal hyperalgesia observed on the ipsirateral side in nerve-ligated mice was significantly suppressed by a single pre-microinjection of a glial-modulating agent propentofylline into the CG 24 h before nerve ligation. These results suggest that chronic painful stimuli induces astrocyte activation in the CG, whereas they do not affect the cell proliferation/differentiation from neural stem cells in the CG and the migration of neural stem cells from the SVZ area. The astrocyte activation in the CG may, at least in part, contribute to the development of a chronic pain-like state following sciatic nerve ligation in mice.

Yokukansan enhances the proliferation of B65 neuroblastoma.

Yokukansan, a traditional Japanese herbal medicine, has been considered to be a novel alternative treatment for several neurological diseases such as neurodegenerative disorders, as well as neurosis, insomnia, and behavioral and psychological symptoms in Alzheimer's disease. Moreover, it has been shown that yokukansan has antidepressant-like and pain-relieving effects in animal models. Recently, several studies have shown that yokukansan has a neuroprotective effect. In this study, we focused on whether or no yokukansan influences cell proliferation related to cell-cycle progression by using B65 neuroblastoma cells derived from monoaminergic neurons. Under treatment with yokukansan, the proliferation rate of B65 neuroblastoma cells significantly increased in a dose-dependent manner. In particular, a proliferative effect was observed after treatment with yokukansan for 48 h and 72 h. Moreover, among seven medicinal herbs that comprise yokukansan, both Bupleuri Radix and Glycyrrhize Radix also enhanced the proliferation of B65 neuroblastoma cells. We assessed the effect of yokukansan on p44/42 mitogen-activated protein kinase (MAPK) phosphorylation in B65 neuroblastoma cells, and found that yokukansan increased p44/42 MAPK phosphorylation after treatment for 48 h. In contrast, neither Bupleuri Radix nor Glycyrrhize Radix altered the level of p44/42 MAPK phosphorylation, although they did increase cell proliferation. Our findings suggest that yokukansan has a cell-proliferative due to both Bupleuri Radix and Glycyrrhize Radix, and this is unrelated to the p44/42 MAPK signaling cascade.

Neuroprotective effect of liquiritin as an antioxidant via an increase in glucose-6-phosphate dehydrogenase expression on B65 neuroblastoma cells.

Glycyrrhiza (the roots and rhizomes of licorice) has been used worldwide as both an herbal nutraceutical and herbal medicine. In addition, it is well known that Glycyrrhiza contains various compounds with biological effects, such as anti-viral, anti-inflammatory, immunoregulatory, anti-tumor and neuroprotective effects. Among the various compounds in Glycyrrhiza, the active compounds that show biological activity are thought to include glycyrrhizin, glycyrrhetinic acid, glabridin, licochalcones and liquiritin. In the present study, we investigated the biological effects of three of these compounds (glycyrrhizin, liquiritin and isoliquiritin) on B65 neuroblastoma cells derived from serotonergic neurons. Among these three compounds, only liquiritin enhanced the proliferation of B65 neuroblastoma cells. In contrast, both glycyrrhizin and isoliquiritin, particularly at high concentrations had cytotoxic effects. Cells were treated with various cytotoxic agents and liquiritin could ameliorate the cytotoxicity induced by menadione sodium bisulfate in a dose-dependent manner. We also examined the effect of liquiritin on cell survival by evaluating the expression levels of phospho-p44/42 mitogen-activated protein kinase, cyclin-related proteins and glucose-6-phosphate dehydrogenase, which produces nicotinamide adenine dinucleotide phosphate. Under treatment with liquiritin, the protein expression level of glucose-6-phosphate dehydrogenase increased in a dose-dependent manner. In contrast, the protein expression level of cyclin-related proteins did not change at all under treatment with liquiritin. These results suggest that liquiritin, which is contained in Glycyrrhiza, may enhance cell survival by increasing the protein expression level of glucose-6 phosphate dehydrogenase.

Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT).

Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.

Involvement of alpha7- and alpha4beta2-type postsynaptic nicotinic acetylcholine receptors in nicotine-induced excitation of dopaminergic neurons in the substantia nigra: a patch clamp and single-cell PCR study using acutely dissociated nigral neurons.

The receptor subtypes, which mediate nicotine-induced excitation of dopaminergic neurons in the substantia nigra, were investigated by whole-cell patch clamp studies and single-cell RT-PCR using acutely dissociated nigral neurons. Three types of current were observed when acetylcholine (1 mM) was applied to the neurons in the presence of atropine (1 microM) by the U-tube system, which allowed the rapid application of drugs. In 50% of neurons examined, acetylcholine (1 mM) plus atropine (1 microM) evoked a current with a rapidly desensitizing decay phase (designated as type Ia current). In 14% of neurons tested, the current induced by acetylcholine plus atropine had a decay phase with slow desensitization (designated as type II current). The third type of response, which had both characteristics of type Ia and II currents, was evoked in 36% of neurons tested (designated as type Ib currents). Nicotine (1 mM) also induced three types of inward currents which were similar to those induced by acetylcholine (1 mM) plus atropine (1 microM). In all three types of current, nicotine (0.1 microM-1 mM)-evoked inward currents were dose-dependent. Type Ia and II currents were inhibited by methyllycaconitine (MLA, 0.01 microM), a selective nicotinic alpha7 receptor antagonist, and dihydro-beta-erythroidine (DHbetaE, 0.1 microM), an antagonist for alpha4beta2 receptor, respectively. In type Ib currents, a fast-decaying phase was inhibited by MLA (0.01 microM), while a slow-decaying phase was blocked by DHbetaE (0.1 microM). After recording the type Ib current, single-cell RT-PCR analysis was performed using aspirated cytoplasm as total RNA templates. The results revealed that mRNAs for alpha7 nicotinic receptor subunit and tyrosine hydroxylase were detected in the same single neuron tested, which confirms the existence of alpha7-type nicotinic acetylcholine receptor in dopaminergic neurons of this area. These results suggest that nicotine directly acts on postsynaptic alpha7- and alpha4beta2-type nicotinic acetylcholine receptors and induces inward current, which result in the excitation of dopaminergic neurons in the substantia nigra.