Kazuo Washiyama

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.

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

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. 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 A1 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. 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. The GIRK currents induced by ethanol were also attenuated in the presence of fluoxetine. 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.

Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry.

Using polymerase chain reaction-assisted single-strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, mutations in the p53 tumor suppressor gene were examined in 19 low-and high-grade gliomas. By PCR-SSCP and nucleotide analyses, p53 gene mutation was seen in 7 gliomas. Out of the 7 mutations, 3 were located at the CpG site of the previously proposed hot-spot codons 248 and 273, 2 were at codons 171 and 214 and the other 2 were in intron 5, 1 at the splice acceptor site and the other in the vicinity of the splice donor site. The latter 4 mutations have not, or only rarely, been observed in gliomas or in other tumors. However, their effect on the structural and functional alteration of the p53 protein was suggested by positive intranuclear p53 immunostaining in neoplastic cells in 3 mutations including the 1 at the splice acceptor site. In connection with glioma grading, the p53 gene mutation was shown to have occurred in both low- and high-grade gliomas, often in most of the neoplastic cells, as suggested by lack of distinct normal bands and ladders in SSCP and direct sequencing, respectively. The absence of recurrence and malignant transformation over a considerably long postoperative time in our low-grade glioma cases suggested that the p53 gene mutation might not be sufficient for the progression from low- to high-grade gliomas. The frequency of detection of mutation was 7/19(37%) by PCR-SSCP, 8/19(42%) by immunohistochemistry and 10/19(53%) by both methods. The results of PCR-SSCP and immunohistochemistry were consistent in 14 cases(73.7%), but not in 5 cases(26.3%). Thus, the use of both methods was recommended to survey the occurrence of p53 gene mutation more accurately.

Immunohistochemical analysis of tumor-infiltrating lymphocytes and major histocompatibility antigens in human gliomas and metastatic brain tumors

A subpopulation of tumor-infiltrating lymphocytes (TILs) and the expression of major histocompatibility complex (MHC) antigens of the tumor cells were examined in 14 glioma and 13 metastatic brain tumor tissues. In both tumors, most of the TILs were T lymphocytes, and both phenotypes of the cytotoxic/suppressor and helper/inducer T lymphocyte were found. On examination of MHC antigens, beta 2-microglobulin was shown intensely on tumor cells in all cases, and the monomorphic determinant of the human leukocyte antigen (HLA-DR) was shown in 10 glioma and in 5 metastatic cases. The correlation between the number of TILs and MHC antigen expression on tumor cells was equivocal as a whole in cases of both glioma and metastatic brain tumor.

Clinicopathological study of pineal parenchymal tumors: correlation between histopathological features, proliferative potential, and prognosis

This study describes the clinicopathologic features of 13 cases with pineal parenchymal tumors. Based on the histopathologic findings, especially the extent of atypia and pineocytic differentiation as determined by Bodians staining, we classified these tumors into pineocytomas (4), pineocytomas with anaplasia (4) and pineoblastomas (5). All the cases with pineocytoma and pineocytoma with anaplasia were adults, and all the cases with pineoblastoma were younger children. One patient with pineocytoma died of other disease 7 months after initial treatment. One patient with pineocytoma with anaplasia died 168 months after initial treatment. The other patients with pineocytoma and pineocytoma with anaplasia survived between 9 and 179 months after surgery. However, all of the five pineoblastoma patients died within 14 months after initial treatment. The mean MIB-1 index in pineoblastomas was significantly higher than that in other types of pineal parenchymal tumors, but there were no differences between pineocytomas and pineocytomas with anaplasia with respect to the MIB-1 index. The mean MIB-1 index in neurofilament protein-immunopositive cases was significantly lower than that in immunonegative cases. With regard to the malignant potential, we emphasize that a clear distinction should be made between pineoblastomas in children and other types of pineal parenchymal tumors in adults.

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 α1 adrenergic, N-methyl-D-aspartate, serotonin and σ 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 κ-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.

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

SummaryNuclear factor (NF)-κB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-κB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-κB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-κB activity. In this study, we investigated inhibitory effects of curcumin on NF-κB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-κB 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 [3H]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-κB 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-κB activity by curcumin.

Inhibitory effects of the antiepileptic drug ethosuximide on G protein-activated inwardly rectifying K+ channels.

Antiepileptic drugs protect against seizures by modulating neuronal excitability. Ethosuximide is selectively used for the treatment of absence epilepsy, and has also been shown to have the potential for treating several other neuropsychiatric disorders in addition to several antiepileptic drugs. Although ethosuximide inhibits T-type Ca(2+), noninactivating Na(+), and Ca(2+)-activated K(+) channels, the molecular mechanisms underlying the effects of ethosuximide have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) channels (GIRK, or Kir3) play an important role in regulating neuronal excitability, heart rate and platelet aggregation. In the present study, the effects of various antiepileptic drugs on GIRK channels were examined first by using the Xenopus oocyte expression assay. Ethosuximide at clinically relevant concentrations inhibited GIRK channels expressed in Xenopus oocytes. The inhibition was concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. However, the other antiepileptic drugs tested: phenytoin, valproic acid, carbamazepine, phenobarbital, gabapentin, topiramate and zonisamide, had no significant effects on GIRK channels even at toxic concentrations. In contrast, Kir1.1 and Kir2.1 channels were insensitive to all of the drugs tested. Ethosuximide also attenuated ethanol-induced GIRK currents. These inhibitory effects of ethosuximide were not observed when ethosuximide was applied intracellularly. In granule cells of cerebellar slices, ethosuximide inhibited GTPgammaS-activated GIRK currents. Moreover, ADP- and epinephrine-induced platelet aggregation was inhibited by ethosuximide, but not by charybdotoxin, a platelet Ca(2+)-activated K(+) channel blocker. These results suggest that the inhibitory effects of ethosuximide on GIRK channels may affect some of brain, heart and platelet functions.

Primary malignant lymphoma of the brain: an immunohistochemical study of eight cases using a panel of monoclonal and heterologous antibodies.

SummaryFrozen sections of eight primary malignant lymphomas of the brain were examined by the immunohistochemical methods using a panel of monoclonal and heterologous antibodies to B lymphocyte (immunoglobulins, BA-1, Leu-12 and HLA-DR), T lymphocyte (OKT-11 and Leu-1) and monocyte (OKM-1) surface markers. Paraffin sections were also used in the examination of cytoplasmic immunoglobulins. Surface and/or cytoplasmic immunoglobulins (Ig) were observed in seven cases,four of which were shown to be distinctly monoclonal and the other three less so. The remaining 1 case showed no distinct staining for Ig. BA-1, Leu-12 and HLA-DR stainings were positive in four, four and five cases, respectively. The marker phenotypes of (BA-1, Leu-12, HLA-DR) were shown to be (+,+,+) in one lymphoma, (+,-,-) in three, (-,+,+,)in three, and (-,-,+) in one. Thus, it was demonstrated that the present lymphoma cases showed a marked immunological heterogeneity, and it was shown that all of them including the Ig-negative case revealed one or more of these three additional B cell markers, indicating B cell lineage of these cases. Examination of T cell and monocyte markers revealed positive staining in normal or reactive lymphoid cells distributed around blood vessels or sporadically in tumor tissues, but not in lymphoma cells. Epstein-Barr virus (EBV)-associated nuclear antigen was not demonstrated in the seven cases examined, making it unlikely that these lymphomas were related with EBV infection.

Neuroendocrine markers in central nervous system neuronal tumors (gangliocytoma and ganglioglioma)

SummaryWe studied five cases of central nervous system neuronal tumor, one gangliocytoma and four gangliogliomas, both ultrastructurally and immuno-histochemically, using antibodies to neuroendocrine markers including tyrosine hydroxylase (TH), serotonin (5HT), somatostatin (SOM), met-enkephalin (MEK), leu-enkephalin (LEK), substance P (SP), gastrin, vasopressin, oxytocin, vasoactive intestinal polypeptide, adrenocorticotropic hormone and calcitonin. In all cases, the presence of dense-core vesicles (60–250 nm) in the neuronal elements was the characteristic ultrastructural finding. Synapses were observed in two cases. Immunohistochemically, variable numbers of neuronal cells showed positive staining for SOM in five cases, TH, MEK and LEK in three cases, and 5HT and SP in one case each. The others were negative. Positive immunoreactivity for multiple markers was shown in all cases. SOM, TH, 5HT and SP were present in the small- to medium-sized cells, while MEK and LEK were almost exclusively confined to the large cells. Our study clearly indicated that these tumors contained neuronal cells which were not homogeneous with regard to neuroendocrine markers.