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Kazuo Washiyama

Publications and source records attributed to Kazuo Washiyama.

14 recordsLinked to original sources
PubMed2006

Inhibition of G protein-activated inwardly rectifying K+ channels by the antidepressant paroxetine.

Paroxetine is commonly used as a selective serotonin reuptake inhibitor for the treatment of depression and other psychiatric disorders. However, the molecular mechanisms of the paroxetine effects have not yet been sufficiently clarified. Using Xenopus oocyte expression assays, we investigated the effects of paroxetine on G protein-activated inwardly rectifying K+ (GIRK) channels, which play an important role in reducing neuronal excitability in most brain regions and the heart rate. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2, or GIRK1/GIRK4 subunits, paroxetine reversibly reduced inward currents through the expressed GIRK channels. The inhibition was concentration-dependent, but voltage-independent and time-independent during each voltage pulse. However, two structurally different antidepressants: milnacipran and trazodone, caused only a small inhibition of basal GIRK currents. Additionally, Kir1.1 and Kir2.1 channels were insensitive to all of the antidepressants. Furthermore, the GIRK currents induced by activation of A1 adenosine receptors or by ethanol were inhibited by extracellularly applied paroxetine in a concentration-dependent manner, but not affected by intracellularly applied paroxetine. Our results suggest that inhibition of GIRK channels by paroxetine may contribute partly to some of its therapeutic effects and adverse side effects.

Adrenergic Uptake Inhibitors↗
PubMed2006

Inhibition of G protein-activated inwardly rectifying K+ channels by ifenprodil.

G protein-activated inwardly rectifying K+ channels (GIRK, also known as Kir3) are regulated by various G-protein-coupled receptors. Activation of GIRK channels plays an important role in reducing neuronal excitability in most brain regions and the heart rate. Ifenprodil, which is a clinically used cerebral vasodilator, interacts with several receptors, such as alpha1 adrenergic, N-methyl-D-aspartate, serotonin and sigma receptors. However, the molecular mechanisms underlying the various clinically related effects of ifenprodil remain to be clarified. Here, we examined the effects of ifenprodil on GIRK channels by using Xenopus oocyte expression assays. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, ifenprodil reversibly reduced inward currents through the basal GIRK activity. The inhibition was concentration-dependent, but voltage- and time-independent, suggesting that ifenprodil may not act as an open channel blocker of the channels. In contrast, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to ifenprodil. Furthermore, GIRK current responses activated by the cloned kappa-opioid receptor were similarly inhibited by ifenprodil. The inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly, and were not affected by extracellular pH, which changed the proportion of the uncharged to protonated ifenprodil, suggesting its action from the extracellular side. The GIRK currents induced by ethanol were also attenuated in the presence of ifenprodil. Our results suggest that direct inhibition of GIRK channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects.

Adrenergic alpha-Antagonists↗
PubMed2006

Gene expression in the brain from fluoxetine-injected mouse using DNA microarray.

Previously we have examined the effects of phencyclidine and clozapine upon the gene expression in the mouse brain. Recently, fluoxetine (Prozac) has been introduced for the therapeutic purpose as an antidepressant drug. Miledi et al. reported blockage of mouse muscle and neuronal nicotinic acetylcholine receptor by various concentrations of fluoxetine. Furthermore, Kobayashi et al. discovered that fluoxetine inhibits G protein activated inwardly rectifying G protein activated K(+) (GIRK) channels using Xenopus oocyte expression assay. From these experiments, we considered that it might be interesting to study the effects of fluoxetine on the gene expression in the mouse brain. After we have injected fluoxetine once a day into mouse for 20 days, we sacrificed mouse by decapitation and extracted RNA from mouse cerebral cortex. We used DNA microarray method for examining the gene expression in the brain. We found the downregulation of many spot signals in the fluoxetine-treated mouse, for example cholecystockinin and prostaglandin D2 synthase.

Animals↗
PubMed2005

Inhibition of cellular proliferation and induction of apoptosis by curcumin in human malignant astrocytoma cell lines.

Nuclear factor (NF)-kappaB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-kappaB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-kappaB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-kappaB activity. In this study, we investigated inhibitory effects of curcumin on NF-kappaB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-kappaB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [(3)H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-kappaB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-kappaB activity by curcumin.

Antineoplastic Agents↗
PubMed2005

Delayed maturation of neuronal architecture and synaptogenesis in cerebral cortex of Mecp2-deficient mice.

We detected morphologic abnormalities in the cerebral cortex of Mecp2-hemizygous (Mecp2(-/y)) mice. The cortical thickness of both somatosensory and motor cortices in mutants did not increase after 4 weeks of age, as compared with that in wild-type male mice. The density of neurons in those areas was significantly higher in layers II/III and V of Mecp2(-/y) mice than in wild-type mice, particularly in layers II/ III after 4 weeks of age. In layer II/III of the somatosensory cortex of Mecp2(-/y) mice, the diameter of the apical dendrite was thin and the number of dendritic spines was small. Electron microscopy revealed that two-week-old mutants already had numerous premature postsynaptic densities. These results indicate that Mecp2(-/y) mice suffered delayed neuronal maturation of the cerebral cortex and that the initial neuronal changes were caused by premature synaptogenesis. Rett syndrome patients with a heterozygous mutation of Mecp2 display developmental disorders including cortical malfunctions such as mental retardation, autism, and epilepsy. Our results provide evidence of the similarity with Rett syndrome brains in some respects and suggest that MeCP2/Mecp2 plays some role in synaptogenesis.

Age Factors↗
PubMed2005

Primary malignant lymphoma of the brain: frequent abnormalities and inactivation of p14 tumor suppressor gene.

Ten primary central nervous system lymphomas (PCNSL, brain lymphomas) were examined for p14 gene exon 1beta deletion, mutation and methylation by Southern blot analysis, nucleotide analysis of polymerase chain reaction clones and Southern blot-based methylation assay. In Southern blot analysis, from the signal densities of the hybridized bands and their similarities to those of exons 2 and 3 in our previous quantitative study, we found that exon 1beta was homozygously deleted in four cases, hemizygously deleted in five cases and not deleted in one case. Thus, the same deletion patterns covered the entire p14 gene for all cases except for one case, which suggested the hemizygous deletion of exons 1beta and 2 and homozygous deletion of exon 3. In addition, although exon 1beta mutation is rare in various tumors, we detected a missense mutation (L50R) in one case with a hemizygous deletion. Methylation of the 5'CpG island of the p14 gene was not suggested for any case without homozygous deletion. Our observation of frequent p14 gene abnormalities (90%) and inactivation (40-60%) was in striking contrast to the same pathological subtype of systemic lymphoma in which p14 gene abnormalities and inactivation were infrequent, suggesting a difference in carcinogenesis between PCNSL and systemic lymphoma.

Adult↗
PubMed2004

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes.

Interferon-alpha (IFNalpha) affects the opioid system. However, the direct action of IFNalpha on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K+ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNalpha on cloned mu-, delta- and kappa-opioid receptors. In oocytes co-injected with mRNAs for either the delta- or kappa-opioid receptor and for GIRK channel subunits, IFNalpha at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNalpha induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the mu-opioid receptor and GIRK channels, IFNalpha had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNalpha, indicating no significant antagonism of IFNalpha toward the opioid receptors. Furthermore, IFNalpha had little or no effect on the mu/delta-, delta/kappa- or mu/kappa-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNalpha weakly activates the delta and kappa-opioid receptors. The direct activation of the delta- and kappa-opioid receptors by IFNalpha may partly contribute to some of the IFNalpha effects under its high-dose medication.

Animals↗
PubMed2004

Inhibition of G protein-activated inwardly rectifying K+ channels by various antidepressant drugs.

G protein-activated inwardly rectifying K+ channels (GIRK, also known as Kir3) are activated by various G protein-coupled receptors. GIRK channels play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate. Modulation of GIRK channel activity may affect many brain functions. Here, we report the inhibitory effects of various antidepressants: imipramine, desipramine, amitriptyline, nortriptyline, clomipramine, maprotiline, and citalopram, on GIRK channels. In Xenopus oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, the various antidepressants tested, except fluvoxamine, zimelidine, and bupropion, reversibly reduced inward currents through the basal GIRK activity at micromolar concentrations. The inhibitions were concentration-dependent with various degrees of potency and effectiveness, but voltage- and time-independent. In contrast, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to all of the drugs. Furthermore, GIRK current responses activated by the cloned A1 adenosine receptor were similarly inhibited by the tricyclic antidepressant desipramine. The inhibitory effects of desipramine were not observed when desipramine was applied intracellularly, and were not affected by extracellular pH, which changed the proportion of the uncharged to protonated desipramine, suggesting its action from the extracellular side. The GIRK currents induced by ethanol were also attenuated in the presence of desipramine. Our results suggest that inhibition of GIRK channels by the tricyclic antidepressants and maprotiline may contribute to some of the therapeutic effects and adverse side effects, especially seizures and atrial arrhythmias in overdose, observed in clinical practice.

Animals↗
PubMed2004

Modulators of G protein-activated inwardly rectifying K+ channels: potentially therapeutic agents for addictive drug users.

G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in the inhibitory regulation of neuronal excitability in most brain regions and heart rate through activation of various G protein-coupled receptors, such as opioid, cannabinoid, and D2 dopamine receptors. Therefore, modulators of GIRK channels may affect many brain functions. We have shown using Xenopus oocyte expression assays that ethanol directly activates GIRK channels, whereas various antipsychotics (thioridazine, clozapine, pimozide, and haloperidol) inhibit the channels. Here we investigated not only the effects of various selective serotonin reuptake inhibitor (SSRI) antidepressants (fluoxetine, citalopram, fluvoxamine, and zimelidine) and risperidone, an atypical antipsychotic, on GIRK channels, but also those of the various drugs tested on other Kir channels using the Xenopus oocyte system. Fluoxetine inhibited GIRK channels, whereas the other SSRIs and risperidone had a small or no effect on the channels. In contrast, Kir1.1 and Kir2.1 channels were insensitive to ethanol and various SSRIs and antipsychotics, although thioridazine weakly inhibited Kir1.1 channels. It has been shown that the function of GIRK channels is involved in seizure susceptibility, antinociception by opioids, cannabinoids, or ethanol, and cocaine reinforcement in studies using GIRK knockout mice and weaver mutant mice that have mutant GIRK2 channels insensitive to G proteins and ethanol. Activation of GIRK channels by opioids, cannabinoids, or ethanol may be one of these key effects. Therefore, GIRK channel modulators might be potential agents for the treatment of users of addictive drugs, such as cocaine, opioids, cannabinoids, and ethanol, as well as for the treatment of epilepsy and pain.

Animals↗
PubMed2003

Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac).

1. The effects of fluoxetine, a commonly used antidepressant drug, on G protein-activated inwardly rectifying K(+) channels (GIRK, Kir3) were investigated using Xenopus oocyte expression assays. 2. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, fluoxetine reversibly reduced inward currents through the basal GIRK activity. The inhibition by fluoxetine showed a concentration-dependence, a weak voltage-dependence and a slight time-dependence with a predominant effect on the instantaneous current elicited by voltage pulses and followed by slight further inhibition. Furthermore, in oocytes expressing GIRK1/2 channels and the cloned Xenopus A(1) adenosine receptor, GIRK current responses activated by the receptor were inhibited by fluoxetine. In contrast, ROMK1 and IRK1 channels in other Kir channel subfamilies were insensitive to fluoxetine. 3. The inhibitory effect on GIRK channels was not obtained by intracellularly applied fluoxetine, and not affected by extracellular pH, which changed the proportion of the uncharged to protonated fluoxetine, suggesting that fluoxetine inhibits GIRK channels from the extracellular side. 4. The GIRK currents induced by ethanol were also attenuated in the presence of fluoxetine. 5. We demonstrate that fluoxetine, at low micromolar concentrations, inhibits GIRK channels that play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate through activation of various G-protein-coupled receptors. The present results suggest that inhibition of GIRK channels by fluoxetine may contribute to some of its therapeutic effects and adverse side effects, particularly seizures in overdose, observed in clinical practice.

Animals↗
PubMed2002

Isolation and structure of the mouse 14-3-3 eta chain gene and the distribution of 14-3-3 eta mRNA in the mouse brain.

14-3-3 protein is a brain-specific protein discovered by Moore and Perez, but at present is thought to be a multifunctional protein. To clarify the brain-specific function of the protein, we intend constructing a 14-3-3 eta gene knock-out mouse. As the first step of this process, we isolated the mouse 14-3-3 eta chain gene and determined its structure. The mouse gene is about 10 kb long and composed of two exons separated by a long intron. The transcription start site was identified and the polyadenylation signals (AATAAA) were found in exon 2 of the mouse gene. In the 5'-upstream sequence, we found several cis elements including a CRE sequence, a TATA box-like sequence, and a C/EBP element. Furthermore, the distribution of 14-3-3 eta mRNA in the mouse brain was examined by in situ hybridization histochemistry. The highest signals were found in the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and the olfactory bulb neurons of the adult mouse. Neuronal expression of 14-3-3 eta in these regions mRNA may generally increase during postnatal brain development. The distribution of protein kinase C gamma in the mouse brain was also examined by immunohistochemistry. From the distribution of 14-3-3 eta mRNA and protein kinase C gamma in the mouse brain, the involvement of these compounds in the induction and maintenance of LTP was discussed.

14-3-3 Proteins↗
PubMed2002

Primary malignant lymphoma of the brain: mutation pattern of rearranged immunoglobulin heavy chain gene.

Using reverse transcription-polymerase chain reaction (RT-PCR), six primary brain lymphomas, pathologically diagnosed as diffuse large B-cell lymphoma, were examined for rearranged VH-D-JH sequences of the immunoglobulin heavy chain gene, focusing on somatic mutations and intraclonal heterogeneity. The reliability of the isolated PCR clones was confirmed by in situ hybridization (ISH) with complementarity-determining region (CDR) 3 oligonucleotide probes. Sequence analysis of the PCR clones revealed a high frequency of somatic mutation, ranging from 8.8 to 27.3% (mean 18.2%) in the VH gene segments in all the lymphomas. A significantly lower frequency of replacement (R) mutations than expected was also seen in their frameworks (FRs) in all cases. These findings suggested that the precursor cells were germinal center (GC)-related cells in these lymphomas. However, despite extensive cloning experiments, intraclonal heterogeneity was not detected in any case except for one in which it could not be ruled out. Thus, it seemed likely that all of our brain lymphomas were derived from GC-related cells and that at least most of them were from post-GC cells.

Aged↗
PubMed2002

Aberrant nuclear factor-kappaB activity and its participation in the growth of human malignant astrocytoma.

OBJECT: It has been suggested that nuclear factor (NF)-kappaB, a pleiotropic transcription factor, controls cell proliferation. The authors examined NF-kappaB activity and its participation in the growth of human malignant astrocytoma. METHODS: The authors examined NF-kappaB activity in human malignant astrocytoma cell lines and high-grade astrocytoma tissues by using electrophoretic mobility shift assays and immunohistochemical studies, respectively. In addition, messenger (m)RNA expression of p50 and RelA, which are representative subunits of NF-kappaB, and IkappaBalpha, which is a representative inhibitory protein of NF-KB, were analyzed using Northern blot hybridization in the astrocytoma cell lines. Furthermore, alterations in DNA synthesis and cell growth in the astrocytoma cell lines were examined after inhibition of NF-kappaB activity by RelA antisense oligodeoxynucleotide. The authors found NF-kappaB activity in all astrocytoma cell lines and high-grade astrocytoma tissues that were examined, but not in the fetal astrocyte strain or in normal cerebral tissue. Expression of p50, RelA, and IkappaBalpha mRNA was found in the fetal astrocyte strain and normal adult brain tissue, in addition to the astrocytoma cell lines. The relative levels of expression of these mRNAs were similar among these cell lines, the cell strain, and normal tissue. The RelA antisense oligodeoxynucleotide specifically reduced the levels of RelA mRNA expression and NF-kappaB activity in the astrocytoma cell lines, thus significantly inhibiting their DNA synthesis and cell growth. CONCLUSIONS: Human malignant astrocytoma cells have aberrant NF-KB activity, which promotes their growth. This activity is not associated with aberrant expression of p50 and RelA.

Age Factors↗