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Volume 108 (1996)
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Volume 108, Issue supplement
Displaying 1-30 of 30 articles from this issue
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Motoya Katsuki1996 Volume 108 Issue supplement Pages 1-2
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSRecently, gene targeting enables site-directed mutagenesis in the mammalian genome. It utilizes homologous recombination between DNA sequences of a targeting vector and the genome of mouse ES cells. Gene targeting conventionally achieves the disruption of the gene, or “gene knock-out”, inserting the drug resistant gene sequence not only as a selected marker, but also as an insertion sequence into the target gene by homologous recombination. Subsequent germ-line transmission of the ES cells through injection chimeras has contributed to the studies on the biological function of the disrupted gene in vivo.
Biological studies to create new drugs for human diseases could be furthermore elaborated if we were able to replace a target gene with human homologue. For this purpose, we have devised a new method that is able to replace the target gene with any sequences constructed in the replacement vector; including subtle mutations, deletions, insertions, human homologues, or totally non-homologous fragments. We conclude that this method, “universal gene replacement”, now allows us to alter any part of mammalian genome as desired DNA sequences of the target locus and it could be developed to a potent method for the generation of the humanized animal models.View full abstractDownload PDF (1552K) -
Kinya Kuriyama, Masaaki Hirouchi1996 Volume 108 Issue supplement Pages 3-8
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSThe GABA receptors are classified into two major subtypes, GABAA and GABAB. The many inhibitory central acting drugs such as benzodiazepines and barbiturates affect the GABAA receptors. The GABAA receptors consist of several subunits and form C1 channel. The multiple combinations of subunits exhibit varieties of pharmacological actions as GABAA and benzodiazepine receptors. On the other hand, the GABAB receptor, one of metabotropic receptors, is a 80 kDa protein coupled with Gi families and the presence of subtypes in the GABAB receptor is proposed from various biochemical or pharmacological studies. The therapeutic applications of GABAB receptor agonists and antagonists are attempted in the cognitive disorders, epilepsy, depression and pain.View full abstractDownload PDF (5208K) -
Kousaku OKUBO1996 Volume 108 Issue supplement Pages 9-12
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSExpression profile is a list of genes acting in a tissue. It is made by measuring the occurence of each transcript by random sequencing of clones from 3'-directed cDNA library made from a tissue. Since 3'-directed libraries faithfully represent the molecular composition in material mRNAs, the occurence of any GS reflects the abundance of corresponding transcript in the source tissue.
BodyMap is made by compiling Expression Profiles of various tissues. In the BodyMap, the expression strength of genes, known or novel, are given as a function of tissues or cell types. At present, we have collected expression information of over 12000 genes among 43 different tissues.
Using this database, tissues can be characterized by their gene expression and genes can be categorized by their expression pattern. Tissue specific, hosekeeping, characteristic to cancers, any sort of genes can be selected from the BodyMap by in silico experiments.View full abstractDownload PDF (2644K) -
Nobuaki Yoshida1996 Volume 108 Issue supplement Pages 13-17
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSNF-IL6 is a basic-leucine zipper transcriptional factor and belongs to the C/EBP family. To investigate the role of NF-IL6 in vivo, we have generated NF-IL6 deficient mice by gene targeting. NF-IL6 deficient mice were highly susceptible to infection with Listeria monocytogenes, and all died within five days after intraperitoneal injection of only 500cfu of bacteria. Electron microscopic observation revealed the escape of a large number of pathogens from the phagosome to the cytoplasm in activated macrophages from NF-IL6 deficient mice. However, cytokines involved in macrophage activation, such as TNF and IFNγ, were induced normally in NF-IL6 deficient mice. Nitric oxide (NO) was produced to the similar extent in macrophages from both wild-type and NF-IL6 deficient mice. These data demonstrated that NF-IL6 is essential for macrophage bactericidal activity and there exists NO-independent mechanism of that activity.
NF-IL6β, which also belongs to the same family and are induced similarly upon inflammation. However, NF-IL6β deficient mice were resistant to the Listeria infection. This could be explained by the redundant function by NF-IL6.View full abstractDownload PDF (3472K) -
Akira Murakami1996 Volume 108 Issue supplement Pages 18-23
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSTranslational inhibition by oligonucleotides complementary to mRNA leads us to the new field of the gene therapy. The concept, so called antisense strategy, has been applied to cure several incurable diseases such as HIV infection and cancer, and moreover to study functions of proteins and genes. Some of those trials are very promising and in the Phase II status. On the other hand, these days, several problems have been critically cited. They are mainly concerning with the mechanism of antisense effects. How do they penetrate into cells? How do antisense DNAs interact with target genes? How do they interact with endogenous proteins? Are there any non-antisense mechanism? Those problems must be solved before the strategy is to be used to patients. This review deals with the detailed evaluation of such problems and with the future prospects of the antisense strategy.View full abstractDownload PDF (4672K) -
Naofumi Mukaida, Kouji Matsushima1996 Volume 108 Issue supplement Pages 24-29
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSAccumulating evidence indicates that members of a novel supergene family, chemokines, play a central role in the establishment of various types of inflammatory reactions by recruiting and activating a specific type(s) of leukocytes. We have established the essential role of interleukin-8 in several types of neutrophil-mediated acute inflammatory models in rabbits by injecting a highly potent neutralizing monoclonal antibody to IL-8. Here, we discussed our two approaches to develop a novel type of anti-inflammatory agents targeting IL-8; humanization of the monoclonal antibody by complementary determining region grafting method and identification of an inducible IκBα protein kinase which is essentially involved in NF-κB-mediated IL-8 gene transcription.View full abstractDownload PDF (4672K) -
Keizo Inoue1996 Volume 108 Issue supplement Pages 30-34
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSIntracellular transport of cholesterol was monitored with mouse penitoneal macrophages. Efflux of endocytosed cholesterol from lysosome is dependent on pH gradient between lysosomal membranes iand sensitive to C 17 or 20 oxosteroids. The “transport” of cholesterol through lysosomal membranes is specific to cholesterol structure. These indicate that there are some cell machinaries which are rerponsible for intracellular traffics of cholesterol.
We could detect α-tocopherol specific binding protein (transfer protein) in liver cytosolic fraction. This protein stimulates the transfer of α-tocopherol between membranes. The defficiency of this protein causes severe shortage of α-tocopherol in body, resulting in ataxia. This indicates that the protein detected in liver is responsible for recycling of α-tocopherol in body.View full abstractDownload PDF (3499K) -
Masashi SASA, Taku AMANO, Hiroaki MATSUBAYASHI, Kumatoshi ISHIHARA, Ko ...1996 Volume 108 Issue supplement Pages 35-40
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSEpileptic model animals, Spontaneously epileptic rat (SER) and Noda epileptic rats (NER), were examined to determine if they were useful for the evaluation of acute and chronic antiepileptic drugs. SER is a double mutant (zi/zi, tm/tm) exhibits tonic convulsion and absence-like seizures. The antiepileptic profile of a single application of conventional drugs in SER was similar to those found in human epilepsy. Novel antiepileptics such as topiramate, clobazam, and piracetam were successfully evaluated with SER for their anticonvulsant effects. The antiepileptic effects of repeated administrations of novel drugs could also be evaluated with this animal. The long-lasting effects of the antiepileptics were similar to those in human epilepsy. In SER hippocampal slices, long-lasting depolarization shift accompanying repetitive firing obtained in CA3 pyramidal cells with a single stimulation of the mossy fibers was inhibited by topiramate, but the firing induced by applying a depolarizing pulse in the cell was not affected, suggesting that the drug interferred with synaptic transmission in the mossy fiber-CA3 system of SER. Thus SER can also be empolyed for elucidating the mechanisms underlying the antiepileptic effects of the drug in question. Recently, a novel epileptic model animal NER, was developed. The clinical features resemble the tonicclonic seizures in human epilepsy. Multiple high voltage spikes appear in cotical EEG during seizures. The audiogenic seizures were induced by acoustic priming of a bell sound which had been given weekly to the NER since 3 weeks of age. This audiogenic seizure was inhibited by the conventional antiepileptics. Thus, both SER and NER are useful models for evaluating the acute and chronic antiepileptic effects of novel drugs.View full abstractDownload PDF (4870K) -
Kazuo UMEMURA1996 Volume 108 Issue supplement Pages 41-46
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSIn this study, the thrombotic occlusion of the middle cerebral artery (MCA) was induced by photochemical reaction between Rose Bengal and green light which causes endothelial injury followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of photochemical reaction. Using this model, we have investigated effects of antithrombotic agents, thrombolytic agents and neuroprotective agents. A thromboxane A2 receptor antagonist, ADP-induced platelet aggregation inhibitor and a selective thrombin inhibitor-inhibited the MCA thrombogenesis, suggesting that platelets play a key role in MCA thrombosis in this model. A tissue-type plasminogen activator (tPA), administered intravenously 30 min after the MCA occlusion could induce reopening of the thrombotically occluded artery, resulting in reduction of cerebral infarction size 24 h after the tPA administration. A glutamate release inhibitor and AMPA/kainate receptor antagonist, administered after the MCA occlusion reduced the size of cerebral infarction 24 h after the MCA occlusion. Using a microdialysis technique, glutamate concentration in the ischaemic border zone was measured and glutamate release was induced following cerebral ischaemia. This suggests that an increase in glutamate concentration in ischaemic border zone may play a key role in the development of cerebral infarction.View full abstractDownload PDF (4799K) -
Yasuo Watanabe, Yusuke Hori, Hiroyuki Utsumi1996 Volume 108 Issue supplement Pages 47-52
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSIn order to investigate the novel treatment for neuro-psychiatric diseases which are caused by the neuronal dysfunctions, we newly developed an animal model which striatal dopaminergic (DA) neuronal system was markedly potentiated by a Ca2+ channel activator After administration of Bay K 8644 (BAY), a L-type Ca2+ channel activator, into rat caudate putamen, the increases of spontaneous locomotor activity (SLMA) and rearing appearences induced by a novel circumstance were significantly enhanced. These increases were due to the extraordinary efflux of DA in caudate putamen after administration of BAY. Pre-treatment of D1 and D2 antagonists markedly inhibited these increases of SLMA and rearing, although these treatments did not effect on the magnificent increase of DA efflux. Pre-treatment of D1 agonist, a dosage of larger 50 nmol of SKF 81297 (SKF), killed BAY treated rats. The smaller dosages of SKF, however, did not influence to the behavioral and biochemical changes seen after the treatment of BAY. Meanwhile, pre-treatments of bromocriptine and talipexole, significantly blocked BAY induced behavioral and biochemical changes, although single administration of each D2 agonist increased SLMA and rearing number, but not the DA efflux. These results suggest that the antagonistic mechanisms of D2 agonists on BAY-induced DA efflux and SLMA potentiation is due to their stimulating to autoreceptor. In conclusion, this animal model is useful for screening D2 agonists which have the anti-Parkinson, anti-schizophrenia and/or neuroprotective action.View full abstractDownload PDF (5011K) -
Toru NISHIKAWA, Asami UMINO, Daisuke SATO, Koyuki KANEDA, Katsunobu TA ...1996 Volume 108 Issue supplement Pages 53-58
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSTo obtain an insight into the possible pharmacotherapy for neuroleptic-resistant symptoms of schizophrenia, we have investigated pharmacological profiles of phencyclidine(PCP)-induced abnormal behavior and c-fos gene expression in the brain using rats because the noncompetitive NMDA receptor blocker PCP elicits schiophreniform psychosis consisting of not only neuroleptic-responsive positive but also neurolepticresistant negative symptoms. Like PCP psychosis, hyperlocomotion, stereotypy and ataxia induced by PCP have been reported to be partially attenuated by neuroleptic drugs in the rat. Intraventricular injection of the D-serine and D-alanine, which are selective agonists for the glycine modulatory site of the NMDA type glutamate receptor, antagonized the ability of PCP to induce abnormal behavior in a stereoselective and a glycine site antagonist-reversible manner. Systemic administration of PCP also resulted in c-fos gene expression, a useful marker of neuronal activity, in various areas of the rat brain in a haloperidol-resistant fashion. In contrast, the neuroleptic drug inhibited the induction of brain c-fos methamphetamine and cocaine that elicit schizophreniform positive symptoms. The distribution patterns of c-fos after PCP were similar to those of a selective NMDA antagonist but different from those of dopamine agonists. The present findings are consistent with the view that disturbed neurotransmission via the NMDA receptor might, at least in part, be involved in the pathophysiology of PCP psychosis and schizophrenia and that PCP-induced behavioral changes and c-fos expression could be appropriate indices for screening a novel class of antipsychotic drugs possessing therapeutic efficacy against neuroleptic-resistant symptoms of these psychotic states.View full abstractDownload PDF (5970K) -
Hideo Saji1996 Volume 108 Issue supplement Pages 59-64
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSInformation on the delineation of the sites and mechanisms of drug action is fundametal to drug discovery and development in the central nervous system. Because this information can be obtained noninvasively, nuclear medical imaging using molecules labeled with gammma-emitting radionuclide is advantageous. Furthermore, since repetive evaluation of the information in the same object can be achieved, this method can also promptly nullify variations within individual body systems. In nuclear medical imaging system, there have recently been a rapid increase in technological advances and the high-resolution system for animals have developed. The application of the system to basic research will provide a leap ininformation on the drug action in the brain. The most straightforward approach is to radiolabel a drug and then to trace its anatomical distribution and binding in the brain. Another approach is to study how the unlabeled drug influences the physiological function measured with a radiolabeled compound (blood flow, glucose metabolism, etc.) and inhibits specific radioligand binding. The use of nuclear imaging system will demonstrate quantitative relationships between drug binding in vivo and drug effects and validate targets and mechanism for drug action in the brain.View full abstractDownload PDF (4306K) -
Nobuhiro NISHIGAKI, Manabu NEGISHI, Atsushi ICHIKAWA1996 Volume 108 Issue supplement Pages 65-69
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSProstaglandin (PG) E receptors are devided into four subtypes. The EP1 and EP3 receptors are coupled to Ca2+ mobilization and the inhibition of adenylate cyclase, respectively, and the EP2 and EP4 receptors are coupled to the same signal transduction pathway, stimulation of adenylate cyclase. To identify the functional differences between EP2 and EP4 receptors, we examined agonist-induced desensitization of these two receptors using Chinese hamster ovary cells, which stably express these receptors. The EP4 receptor underwent short term agonist-induced desensitization, but no such desensitization was observed for the EP2 receptor. In contrast, the EP2 and EP4 receptors displayed similar patterns of down-regulation in response to prolonged exposure to PGE2. On the other hand, PGE2 is rapidly metabolized to 15-keto-PGE2 and subsequently to 13, 14-dihydro-15-keto-PGE2. Thus, we compared the sensitivities of the two receptors to these two metabolites. The EP4 receptor markedly lost the response at the first metabolism, while the EP2 receptor gradually lost the response according to the degree of metabolism, having higher sensitivity to the first metabolite, 15-keto-PGE2, than the EP4 receptor. Therefore, the physiological significance of EP2 and EP4 may lie in their different sensitivity to agonist-induced short term desensitization, and their differential susceptibility to the metabolic inactivation of the agonist.View full abstractDownload PDF (4411K) -
Takuya Kobayashi, Michitaka Kiriyama, Masakazu Hirata, Fumitaka Ushiku ...1996 Volume 108 Issue supplement Pages 70-74
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSWe have cloned eight types and subtypes of the mouse prostanoid receptors, and revealed that all of them have seven transmembrane domains, indicating that they belong to the G-protein-coupled, rhodopsin-type receptor superfamily. Although the overall homology among the prostanoid receptors is not high, there are several regions specifically conserved among them. These regions are considered to form the binding pocket for the structure common to prostanoid molecules, and the other regions to participate in the formation of binding sites which recognize the specific structure for each prostanoid molecule. The chimeric receptors, in which a region of one prostanoid receptor is replaced by the corresponding sequence from the region of another, present useful models for elucidating these ligand binding domains. The PGl2 and PGD2 receptors have relatively high homology (40%) and the same signalling pathway, which we considered prerequisite features for derived chimeric receptors to be stable and functional. The chimeric receptors of the PGl2 and PGD2 receptors were expressed in COS-7 cells, and their abilities to bind [3H]iloprost, a PGl2 agonist, and [3H]PGD2 were examined. The chimeric receptors, in which the carboxyl-terminal portion from the fourth transmembrane domain of the PGl2 receptor is replaced by the corresponding sequence from the region of the PGD2 receptor, did not affect the affinity of iloprost and did not show the PGD2 specific binding. These findings suggest that the carboxyl-terminal portion from the fourth transmembrane domain is not necessary for iloprost specific binding, and that this region is insufficient for the PGD2 binding.View full abstractDownload PDF (3219K) -
Hitoshi KUROSE, Hideo KIKKAWA, Masafumi ISOGAYA, Taku NAGAO1996 Volume 108 Issue supplement Pages 75-80
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSStructual basis of the β-adrenergic receptor (βAR) to confer the β selectivity is largely unknown. We investigated the binding regions of βAR which is important for the subtype selective binding. TA-2005 is a highly selective β2 agonist with the carbostyril structure. Binding regions of the βAR for TA-2005 was investigated by use of chimeric β1 and β2 receptors as comapared with those of non-selective β agonist isoproterenol. Replacement of transmembrane region 2 (TM2) or TM7 of β2AR with those of β1AR decreased the affinity of TA-2005. Deletion of methoxyphenyl group from TA-2005 molecule lost β2 selectivity. Then it is reasonable to assume that methoxyphenyl group interacts with TM2 and TM7 of β2AR, and contributes to the β2 selectivity. Several β2 selective agonists including formoterol and salmeterol also required TM2 or TM7 for the β2 selective binding. T-0509 is a β1 selective full agonist. In contrast with the binding of β2 selective agonist, selective binding of T-0509 to β1AR required the only TM2 of β1AR. These data suggested that TM2 and TM7 of βAR is important for the subtype selective binding.View full abstractDownload PDF (4335K) -
Norimiti NAKAHATA, Yasushi OHIZUMI1996 Volume 108 Issue supplement Pages 81-86
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSU46619, a thromboxane A2 receptor agonist, caused aggregation and shape change in rabbit platelets. The aggregation, but not shape change, was dependent of external Ca2+. A forty times higher concentration of U46619 was needed for aggregation than shape change. U46619 at low concentrations could cause shape change without affecting the internal Ca2+ concentrations. Therefore, thromboxane A2-mediated shape change is independent of Gq-phospholipase C-Ca2+ pathway. U46619 phosphorylated 125 kDa protein at tyrosine residues within several seconds independent of external Ca2+, corresponding to its shape change. The phosphorylation of 125k Da protein was detected in triton-insoluble (cytoskeleton-rich) fraction. These results suggest that stimulation of a shape change-linked thromboxane A2 receptor results in the phosphorylation of 125 kDa protein which may participate in cytoskeltal reorganization during shape change.View full abstractDownload PDF (5310K) -
Hiroshi TSUNEKI, Katsuya DEZAKI, Hiroshi NOJIMA, Ikuko KIMURA, Masayas ...1996 Volume 108 Issue supplement Pages 87-92
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSFew functional roles of neuronal nicotinic receptors (N-nAChR) are clarified, although N-nAChR have been reported to be highly permeable to Ca2+ than the muscle nicotinic receptor (M-nAChR). We have found new regulatory Ca2+ mobilization, RAMIC (Receptor-Activity Modulating Intracellular Ca2+), at the endplate region in the mouse diaphragm muscles treated with a cholinesterase inhibitor by measuring Ca2+-aequorin luminescences. RAMIC mobilization is enhanced via the activation of protein kinase-A by nerve-derived calcitonin gene-related peptide, and promotes the M-nAChR desensitization via the protein kinase-C activation. The RAMIC mobilization is depressed by a N-nAChR antagonist methyllycaconitine (0.1-1 μM) and by monoclonal antibody to β2 subunit of N-nAChR. Furthermore, β2 and α8-related subunits were identified at murine skeletal muscle endplates either by RT-PCR or by immunohistochemical staining. In isolated single skeletal muscle cells loaded with a fluorescent Ca2+ indicator fluo-3 AM, bath-applied ACh (3 μM) elicits two-phasic elevation of [Ca2+]i (fast and slow components) localized beneath the endplate membranes, by using a real time-confocal laser scanning analysis. Methyllycaconitine selectively inhibits the slow Ca2+ component at the similar concentrations (1-5 μM) used to depress RAMIC. These results demonstrate that N-nAChR coexist with M-nAChR at the muscle endplates, and operates the RAMIC mobilization to promote the M-nAChR desensitization. Thus, the neuromuscular function is regulated by dual nAChR system to avoid the overexcitation.View full abstractDownload PDF (5113K) -
Soichi MIWA, Tetsuya MINOWA, Taijiro ENOKI, Xiao-Feng ZHANG, Yasushi I ...1996 Volume 108 Issue supplement Pages 93-98
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSEndothelin-1 (ET-1) induces persistent vasoconstriction via sustained increase in intracellular free Ca2+ concentrations ([Ca2+]i). Mechanisms of the elevation of [Ca2+]i operating at physiologically low concentrations of ET-1 are controversial. Here we report that both native ETA receptors in vascular smooth muscle cells (VSMCs) and recombinant ETA receptors expressed in mouse fibroblasts (Ltk- cells) are functionally coupled with nonselective cation channel, which is permeable to Ca2+ and blocked by mefenamic acid. The channel is persistently activated by a low concentration of ET-1 (10-10 M) without stimulation of inositol trisphosphates (IP3) formation and mediates sustained vasoconstriction.View full abstractDownload PDF (4551K) -
Ken-Ichi FURUKAWA, Satoshi SASAMURA, Miwa SHIRATORI, Yasushi OHIZUMI1996 Volume 108 Issue supplement Pages 99-103
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSThe importance of the plasma membrane Ca2+-pump in regulating the intracellular Ca2+ concentrations ([Ca2+]i) has been recognized. However, the exact role of the plasma membrane Ca2+ pump in the intracellular Ca2+ metabolism of living cells remains to be elucidated. To answer this query antisense oligodeoxynucleotides based on the sequence of the rat plasma membrane Ca2+ pump (PMCA-1) were used to study the effects of inhibiting expression of this protein on the regulation of [Ca2+]i in cultured vascular smooth muscle cells. Antisense oligodeoxynucleotides targeting a sequence [position +1 to +18] of rat PMCA-1 reduced the expression of the pump in a concentration dependent manner, while mismatch or sense oligodeoxynucleotides had no effects. Cytotoxic effects of antisense oligonucleotides were not observed. Antisense treatment affected not only the resting level of [Ca2+]i but also the magnitude of the increase in [Ca2+]i of vascular smooth muscle cells induced by Ca2+ mobilizing agents. 45Ca efflux from vascular smooth muscle cells mediated by plasma membrane Ca2+ pump was also inhibited by the treatment with antisense oligodeoxynucleotides. These results suggest that reduced expression of the plasma membrane Ca2+ pump (PMCA-1) significantly alters intracellular Ca2+ regulation of vascular smooth muscle cells and that PMCA-1 plays important roles in the regulation of intracellular Ca2+ concentrations of vascular smooth muscle cells both in the resting and stimulated states. Antisense oligodeoxynucleotides targeting a sequence of the pump becomes a powerful tool to investigate its physiological role.View full abstractDownload PDF (4355K) -
Yuhei NISHIMURA, Hiroyasu INADA, Toshio TANAKA1996 Volume 108 Issue supplement Pages 104-108
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSProtein kinase A (PKA), protein kinase G (PKG) and protein kinase C (PKC) have crucial roles in signal transduction of smooth muscle cell. However, little is known about a shift of gene expression when PKA, PKG and PKC are activated in the smooth muscle cells. And the exact pathways of signal transduction from activation of PKA, PKG and PKC to regulation of gene expression in the smooth muscle cells are not well understood. Recently, RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) has been developed as a method to identify and characterize alter gene expression in eukaryotic cells. Fingerprinting of RNA populations was achieved using anarbitrarily selected primer at low stringency for first and second strand cDNA synthesis. In this report, we have characterized forskolin, sodium nitroprusside (SNP) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced alterations of gene expression in cultured rat smooth muscle cells using the RAP-PCR method. After incubation with forskolin, SNP and TPA in cultured rat aortic smooth muscle cells, total RNA was isolated and converted to cDNA using an arbitrary primer. Each three agents induced a variety of differential gene expression. These results suggest that several gene expressions were selectively dependent on each intracellular signaling in cultured rat aortic smooth muscle cells.View full abstractDownload PDF (3425K) -
Toshio MATSUDA, Akemichi BABA1996 Volume 108 Issue supplement Pages 109-114
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSWe have previously demonstrated that the Na+-Ca2+ exchange activity was stimulated by nitric oxide (NO)/cGMP and inhibited by ascorbic acid. The present paper demonstrates that the Na+-Ca2+ exchanger is involved in agonist-induced Ca2+ signalling in cultured rat astrocytes. Astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by L-glutamate, noradrenaline (NA) and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced ionomycin-induced increase in [Ca2+]i. NA-induced Ca2+ signal was also attenuated by S-nitroso-L-cysteine and 8-bromo-cGMP, while it was enhanced by 3, 4-dichlorobenzamil, an inhibitor of the Na+-Ca2+ exchanger. Treatment of astrocytes with antisense, not sense, deoxynucleotides to the sequence encoding the Na+-Ca2+ exchanger enhanced ionomycin-induced increase in [Ca2+]i and blocked the effects of SNP and 8-bromo-cGMP in reducing NA-induced Ca2+ signal. Furthermore, ionomycin-induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na+-Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes.View full abstractDownload PDF (4273K) -
Yoshihisa KITAMURA, Hideaki TAKAHASHI, Yasuji MATSUOKA, Yasuyuki NOMUR ...1996 Volume 108 Issue supplement Pages 115-120
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSTo clarify the expression of inducible nitric oxide synthase (iNOS) in vitro and in vivo brain, we examined the effects of intrahippocampal microinjection with interferon-γ (IFN-γ) plus lipopolysaccharide (LPS) and treatment with IFN-γ and LPS in cultured glial cells. 130-kDa iNOS protein was induced after 12 h by intrahippocampal injection with IFN-γ plus LPS, although 160-kDa nNOS expression was not changed. On the other hand, iNOS mRNA was expressed after 6 h, but the mRNA was degraded rapidly thereafter. The injection with IFN-γ plus LPS caused numerous ameboidal microglia and induced MHC class II. In a part of these ameboidal microglia, iNOS-immunoreactivity was observed. Subsequently, immunoreactivities of iNOS and MHC class II were disappeared over 3 days after injection. These results suggest that the intrahippocampal injection with IFN-γ plus LPS induced iNOS mRNA after 6 h and iNOS protein after 12 h in a part of microglia which are activated to the ameboid type and express MHC class II in vivo brain. On the other hand, the cultivation of microglia caused ameboid type. In addition, the treatment with IFN-γ or LPS alone for 24 h induced 130-kDa iNOS protein mainly in microglia. IFN-γ induced tyrosine-phosphorylation of Jak2-tyrosine kinase and transcription factor STAT1α, but LPS did not. However, both iNOS induction by IFN-γ and LPS were inhibited by tyrosine kinase inhibitor herbimycin A. These results suggest that IFN-γ-induced iNOS induction involves activation of Jak2-STAT1α pathway in ameboidal microglia. On the other hand, LPS-induced expression was mediated by other tyrosine kinases.View full abstractDownload PDF (4134K) -
Yasuyuki NOMURA, Tomohiro MIYASAKA, Itsuko BABA, Takashi UEHARA1996 Volume 108 Issue supplement Pages 121-126
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSWe examined the induction of nitric oxide synthase (iNOS) mRNA and neuronal death after forebrain ischemia by 4 vessel occlusion in rat hippocampus. In addition, we tried to characterize whether or not the delayed neuronal cell death is apoptosis or necrosis by means of TUNEL method and staining of chromatin dye, Hoechst 33258. Nuclei of the CAI pyramidal neurons were positive to TUNEL after ischemic insult, but not at the preceding stage. These results suggest that the delayed neuronal death following forebrain ischemia is apoptosis. Moreover, induction of iNOS mRNA was detected 3 days after ischemic insult in hippocampus. It seems to be a close causality between he neuronal death following transient ischemia and iNOS/NO in the hippocampus. We then found that herbimycin A, a potent tyrosine kinase inhibitor, and aspirin inhibited LPS/IFN-γ-induced iNOS induction in C6 glioma cells. It is interesting to examine whether or not both drugs inhibit brain ischemia-induced neuronal apoptosis. We presented and discussed the mechanism of NO-induced apoptosis using NG108-15 cells too.View full abstractDownload PDF (3962K) -
Yoshimi Misu, Yoshio Goshima, Takeaki Miyamae, Yoko Okumura, Jin-Liang ...1996 Volume 108 Issue supplement Pages 127-132
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSL-DOPA is proposed to be a neurotransmitter of the primary baroreceptor afferents terminating in NITS, whereas GABA inhibits baroreflex within NTS. Thus, if our proposal is truly the case, GABA should tonically inhibit pre and/or post-synaptic DOPAergic functions in NTS. Muscimol 10 and 30 μM, locally perfused via probes, inhibited DOPA release by 50 and 60% during microdialysis of NTS in anesthetized rats, with a peak 2 hr after perfusion. Muscimol (10 μM)-induced inhibition was antagonized by noneffectivel0 μM bicuculline. Bicuculline 30 μM alone increased DOPA release in a similar time course. By microinjections into depressor sites of NTS, DOPA 10-60 ng or GABA 3-300 ng elicited dose-dependent hypotension or hypertension. Cardiodepressor responses to DOPA 30 ng were dose-dependently reduced by prior injections of GABA 3-30 ng, also by nipecotic acid 100 ng, but inversely potentiated by bicuculline 10 ng, when respective regulatory response to GABA-related drug returned to control. Meanwhile, DOPA methyl ester, a competitive DOPA antagonist, did not displace [3H]-GABA binding in brain membrane preparations. When a tonic function of endogenously released DOPA was antagonized by this antagonist 1 μg microinjected 1 min previously, cardiopressor responses to GABA 300 ng were reduced by a half. DOPA 10 nM evoked GABA by 40% in the first 20 min sample. Under inhibition of central AADC by perfusion of NSD-1015 200 μM, DOPA 1-100 nM concentration-dependently evoked GABA by 35-60%. DOPA (10 nM)-induced GABA release was not mimicked by dopamine or D-DOPA 10 nM, but was abolished by Ca2+ deprivation or perfusion of tetrodotoxin 1μM. GABA seems to inhibit tonically via activation of GABAA receptors presynaptic DOPA release and postsynaptic depressor and bradycardic responses to DOPA and to induce hypertension and tachycardia in part via inhibition of tonic postsynaptic cardiodepressor function of endogenously released DOPA within NTS. DOPA seems to evoke by itself endogenous GABA from GABAergic neurons as a compensatory mechanism of baroreflex within NTS. These findings further support the idea that DOPA is a neurotransmitter of the primary baroreceptor afferents terminating in rat NTS.View full abstractDownload PDF (5027K) -
Norio Matsuki, Miwa Toyoda, Hiroshi Saito1996 Volume 108 Issue supplement Pages 133-138
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSIn order to elucidate the role of substance P (SP) in emetic responses, effects of resiniferatoxin (RTX), an ultra-potent capsaicin analogue that releases SP, were investigated in Suncus murinus. RTX induced dose-dependent emesis, and beside vomiting, flat down, increase of respiration rate, tremor and eye twitch were notable behavioral changes at a dose of 100 μg/kg. These effects were transient, and animals became normal 30 to 40 min after the injection. RTX-induced emesis was blocked by 30 mg/kg CP-99, 994 and 10 mg/kg ruthenium red. RTX also showed anti-emetic effects. Animals treated with RTX did not respond to cisplatin, copper sulfate, nicotine nor motion after one hour. The anti-emetic effects of RTX were also inhibited by ruthenium red but not by CP-99, 994. Both emetic and anti-emetic effects of RTX were apparently desensitized. Neonatal treatment with capsaicin completely prevented emetic response to RTX but not to cisplatin, copper sulfate, nicotine and motion. RTX significantly decreased the content of SP in the nucleus tractus spinalis nerve trigemini, the dorsal spinal cord but not in the dorsal vagal complex that is considered as part of emetic pattern generator. These results suggest that SP is involved in emetic responses but its action is complicated.View full abstractDownload PDF (4947K) -
Atsushi MIYAMOTO, Hideyo OHSHIKA1996 Volume 108 Issue supplement Pages 139-142
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSOld C57BL/6J mice showed poorer retention of passive avoidance task (PAT) than young-adult mice (6-month-old). Subcutaneous injection of dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) improved the impaired memory retention of PAT in old mice. DHEAS and PS, major neurosteroids that decreases in blood serum with age, may be of use in treatment of neurodegenerative and memory disorders.View full abstractDownload PDF (3209K) -
Akihiro NAKAMURA, Hirohito SHIOMI1996 Volume 108 Issue supplement Pages 143-148
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSSignal transduction system involved in the mechanism of peripheral mechanical hyperalgesia was investigated. All drugs were administered intradermally into the instep of rat hind-paws. Bradykinin (BK)-induced hyperalgesia was significantly suppressed by NO-cGMP pathway inhibitors, as well as prostaglandins (PGs)-cAMP pathway inhibitors, in the paw-pressure test. Concomitant administration of BK with Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase (A kinase), or Rp-8-Br-cGMPS, an inhibitor of cGMP-dependent protein kinase (G kinase) also abolished BK-induced hyperalgesia. Concomitant administration of 8-Br-cAMP (10 nmol) with 8-Br-cGMP (10 nmol) produced mechanical hyperalgesia synergistically. When administered alone, high dose of 8-Br-cAMP (30 and 100 nmol), but not 8-Br-cGMP, produced significant hyperalgesia. The hyperalgesia induced by high dose of 8-Br-cAMP was significantly attenuated by Rp-8-Br-cGMPS, as well as Rp-cAMPS. This result suggests that cross-activation of G kinase by cAMP also plays some role in the production of hyperalgesia. However, peripheral hyperalgesia induced by chemical mediators (BK, adenosine, serotonin and glutamate) or carrageenan was significantly suppressed by NO-cGMP pathway inhibitors. This suggests that G kinase is mainly activated through the NO-cGMP pathway in inflammatory state. In conclusion, an activation of both cGMP- and cAMP-second messenger system plays an important role in the production of peripherally induced mechanical hyperalgesia.View full abstractDownload PDF (5131K) -
Hideki MORITOKI, Tetsuhiro HISAYAMA, Yuichiro AMANO, Kan KIDA, Takesi ...1996 Volume 108 Issue supplement Pages 149-154
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSBnzoyl-pyrimidopyrimidine derivatives (BZPP) has been demonstrated to alleviate hyperalgesia in experimental model of of peripheral neuropathic pain. Activation of iNOS causes hyperalgesia and that adenosine contributes analgesia. In this connection, we investigated the mechanism of action of BZPP in vasculature as a model system, with reference to adenosine and iNOS induction/NO. BZPP prevented LPS- or IL-1β-primed initiation of L-arginine-induced relaxation and cGMP formation in rat thoracic aorta, and nitrite accumulation of cultured aortic smooth muscle cells. Treatment of rat aortic segments with LPS induced iNOS mRNA in 8 h. BZPP attenuated the expression of LPS-stimulated iNOS mRNA, assessed as iNOS/G3PDH ratio. In addition, BZPP potentiated adenosine-induced negative inotropic response of guinea-pig atria, adenosine-induced inhibition of guinea-pig ileum twitch response and adenosine-induced relaxation of rat aorta. Therefore, it is conceivable that inhibitory effect on iNOS induction and subsequent reduction of NO formation, and potentiating action on adenosine-induced effects may be responsible for the analgesic effect of BZPP.View full abstractDownload PDF (3997K) -
Tadahiko HAZATO, Tadahiro KANAZAWA, Masaaki UEKI, Yukio YAMAMOTO1996 Volume 108 Issue supplement Pages 155-160
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSSpinorphin is an endogenous pain-regulated subustance which modulated an enkephalin-degrading activity. Recently, we found that Spinorphin inhibited 02-generation, exocytosis of β-glucuronidase and collagenase by human neutrophils(PMNs) stimulated with FMLP as an inflammatory mediator. In this study, we examined whether Spinorphin exerts anti-inflammatory effects on λ-Carrageenan-nduced acute inflammation in the mouse air pouch.View full abstractDownload PDF (3228K) -
Nobuyuki FUKUISHI, Masakiyo SAKAGUTI, Yoshiyasu FUKUYAMA, Masaaki AKAG ...1996 Volume 108 Issue supplement Pages 161-166
Published: 1996
Released on J-STAGE: February 06, 2007
JOURNAL FREE ACCESSRat peritoneal mast cells were stimulated to generate superoxide anion (02-) by the addition of compound 48/80 (48/80), a typical histamine releaser. Biphasic elevation of [Ca2+]i was also induced by 48/80. The first phase was inhibited by TMB-8 and the second by diltiazem. And 48/80-induced O2- generation was inhibited by TMB-8, forskolin, isoprenaline, dibutyryl cAMP, KT-5926, a MLCK inhibitor, and manoalide, a phospholipase A2 inhibitor, and enhanced by KT-5720, an A-kinase inhibitor. Our data suggest the followings: (1) 48/80-induced 02- generation may be initiated by an increase of [Ca2+]i via cAMP-dependent protein phosphorylation such as MLCK phosphorylation; (2) the mechanisms of O2- generation in mast cells may be similar to that in neutrophils (NADPH-oxidase system). Epinastine, ketotifen, oxatomide and mequitazine dose-dependently prevented the N-formyl-Met-Leu-Phe and phorbol 12-myristate 13-acetate-induced O2- generation from rat neutrophils, but cromolyn sodium did not prevent it. When membrane and cytosol fractions were incubated with each drug, epinastine, ketotifen and mequitazine prevented O2- generation. On the other hand, when only the membrane fraction was incubated with each drug, ketotifen and mequitazine prevented O2- generation, but epinastine did not. Epinastine may inhibit the NADPH oxidase system through the obstruction of NADPH oxidase-associated cytosol components. In conclusion, our studies suggest that the inhibitory effects on O2- generation may be useful as a new marker in the evaluation of innovative antiallergic drugs.View full abstractDownload PDF (3797K)
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