日本薬理学雑誌
Online ISSN : 1347-8397
Print ISSN : 0015-5691
ISSN-L : 0015-5691
112 巻, supplement 号
選択された号の論文の32件中1~32を表示しています
  • David. M. Jacobowitz
    1998 年 112 巻 supplement 号 p. 1-4
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Neuropeptides have now been localized throughout the nervous system. This talk focuses attention on (1) the interplay of peptides and other neurotransmitter systems in the hypothalamus - median eminence - pituitary gland. The multiplicity of neurochemicals is perceived to be responsible for the integrated control of pituitary hormone releasing factors; (2) the role of neuropeptides in the regulation of cardiovascular function in the hypothalamus-preoptic area. We investigated the effects of discrete intrahypothalamic injections of a variety of peptides on blood pressure and heart rate. We concluded that neuropeptides have a diversity of central cardiovascular actions and that not all areas containing a given peptide respond with cardiovascular change when the peptide is injected. Also, peptide specific actions originating within the same nucleus have been demonstrated, and the same peptide may have different vascular effects in different segments of the same nucleus; (3) the colocalization of neuropeptides with other classical neurotransmitters. We have found modulatory behavioral effects (“boxing”) of combinations of transmitters and peptides injected into the postsynaptic site in the brain.
  • 佐藤 昭夫
    1998 年 112 巻 supplement 号 p. 5-9
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Local metabolites have long been considered to play an important physiological role in regulating regional cerebral blood flow (rCBF). However, the evidence reviewed here emphasizes that the regulation of rCBF by central cholinergic nerves is independent of regional metabolism. Activation of the intra cranial cholinergic fibers originating in the nucleus basalis of Meynert (NBM) and septal complex releases acetylcholine in the cortex and hippocampus, which results in vasodilation and an increase in rCBF in the cortex and hippocampus via activation of both muscarinic and nicotinic acetylcholine receptors. Cutaneous sensory stimulation activates the cholinergic nerves originating from the NBM to enhance rCBF. The increase in rCBF at the defuse cortices during walking appears to include an excitation of this NBM-originating cholinergic vasodilation system. Other various inputs to the NBM may have a similar effects to enhance rCBF via activation of that cholinergic system, provided the stimulation is delivered properly. Thus thecombination of pharmacological and nonpharmacological techniques may provide a balance in our attempts to improve cholinergic replacement therapy.
  • 畠中 寛
    1998 年 112 巻 supplement 号 p. 10-14
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    During development, excess neurons are produced about half of which die. The time of cell death (apoptosis) is limited to the period of formation of synapses with the target cells, and the neurons which fail to obtain sufficient amounts of trophic factor(s) released from the target cells are eliminated. This selection system is considered to be a mechanism to ensure formation of a physiologically relevant neuronal network. Mature neurons which correctly execute their functions, however, undergo apoptosis in response to exogenous toxic stimuli. Such stimuli may be responsible for neurodegenerative diseases. The mechanism underlying cell death has been analyzed using in vitro model systems. In the present communication, we used cultured rat cerebellar granule neurons, in which low potassium concentration (LK+) in the medium induces apoptosis, and this apoptosis is prevented by high concentration of potassium(HK+), BDNF. One of the lipid-modifyingk inases, phosphatidylinositol 3-kinase(PI3-K), is also activated by trophic factors includingn eurotrophins. BDNFa nd highK+ prevented low K+-induced apoptosis via PI3-K. BDNFa lso promotest he survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. The mechanism of neuronal apoptosis induced by oxidative stress using CNS neurons and PC12 cells was investigateda, nd we foundt hat generation of reactive oxygens pecies (ROS) is highlya ssociatedw ithapoptosisH. igh oxygeni nducedn euronala poptosisw, hichw as blocked by protein or RNA synthesis inhibitors. Neurotrophic factors and Bcl-2 preventedt his apoptotic cell death. Exposure to hydrogen peroxide, lipid hydroperoxide or serum deprivation triggered apoptosis associated with increased generation of ROS as determined using a ROS-specific fluorescent probe. In cultured cerebellar granule neurons from 15-day-old wild-type and p53-deficient mice, we examinet he role of p53 in regulating the life and death of CNS neurons. When exposure of γ-ray or bleomycin to neurons died in p53 dependent manner. These neuronal deaths were partially prevented by actinomycin D or cycloheximide. The pycnotic nuclei observed in these dying neurons indicated that cell death occurs via apoptosis. Although there are many evidences that p53 is involved in apoptosis in proliferating cells, it is interesting that p53 is also involved in apoptosis in postmitotic neurons as shown in this study.
  • 澤田 誠
    1998 年 112 巻 supplement 号 p. 15-19
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Microglia, macrophage-like cells in the central nervous system (CNS), are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the CNS degeneration and are one of important cells in the CNS cytokine network to produce and respond to a variety of cytokines. Although little is known about microglia in the normal CNS, it is obvious that they are quickly activated in all acute pathological events including apoptosis, neurodegeneration and inflammation. Activation of microglia in apoptosis is a double-edged response; under severe apoptotic conditions microglia act as scavengers removing tissue debris and inducing apoptosis in damaged cells, whereas in more sbtle injury they exert a surveillance function and might play a protective role. The transformation of resting microglia into full blown phagocytes is strictly regulated. To understand molecular basis of controling mechanisms of microglia in apoptosis, the study will need in vivo models. For such purpose, we developed the brain-targeting gene delivery system using immortalized microglia, which can facilitate investigation into the roles of particular microglial genes in apoptosis and gene therapy of several brain disorders
  • 三浦 正幸
    1998 年 112 巻 supplement 号 p. 20-23
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Oligodendrocytes are myelin forming cells in mammalian central nervous system. About 50 % of oligodendrocytes (OLGs) undergo cell death in normal development. In addition, massive OLG cell deaths have been observed in multiple sclerosis (MS). Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes (OLGs). The addition of TNF-a to primary cultures of OLGs significantly decreased the number of live OLGs in 72 h. Chemical inhibitors Ac-YVAD-CHO (a specific inhibitor of caspase-1-like proteases) enhanced the survival of OLGs treated with TNF-a, indicating that caspase-1-mediated cell-death pathway are activated in TNF-induced OLG cell death. caspase-11 is involved in activation of caspase-1. Oligodendrocytes from CASP-11 deficient mice are partially resistant to TNF-induced cell death. These results suggest that the activation of caspases is crucial in TNF-induced OLG cell death and inhibiton of caspase family may be a novel approach to treat neurodegenerative diseases such as MS.
  • 松田 敏夫, 田熊 一浩, 李 英培, 木村 裕治, 藤田 隆司, 馬場 明道
    1998 年 112 巻 supplement 号 p. 24-27
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Astrocytes, the most abundant glial cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We found that reperfusion of cultured astrocytes after Ca2+ depletion causes delayed cell death and that the Na+-Ca2+ exchanger in the reverse mode is responsible for this Ca2+-mediated cell injury (Ca2+ paradox injury). The Ca2+ paradox injury of cultured astrocytes is considered to be an in vitro model of ischemia/reperfusion injury, since a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue. Furthermore, we demonstrated that heat shock proteins, glutathione and calcineurin inhibitors protected astrocytes against Ca2+ paradox-induced cell toxicity. We also observed DNA fragmentation, a typical apoptotic ladder, 2-3 days after hydrogen peroxide exposure. In addition, laser microscopic observation showed that reperfusion after the exposure to hydrogen peroxide caused nuclear condensation of astrocytes. Hydrogen peroxide-induced cell injury and DNA fragmentation were attenuated by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate, 1, 10-phenanthroline and a caspase 3 inhibitor. These findings suggest that astrocytes are one of the targets for ROS and the oxidative stress-induced delayed death of astrocytes is at leaset due to apoptosis.
  • 伊東 祐之
    1998 年 112 巻 supplement 号 p. 28-31
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    It is generally considered that dominatn excitatory control of airway and gastrointestinal tract is exerted by the parasympathetic nervous system, and the transmitter output from the nerve terminals, mainly acetylcholine (ACh), is modulated in many ways for example by ACh itself, prostaglandins E series (PGE) neuropeptides and nitric oxide (NO). These modulations are probably due to the suppression of high-voltage-activated calcium channels in the nerve terminals, since, for example, ACh or prostaglandin E1 & E2 selectively suppressed both the N- and R-type Ca2+ currents, through M2 muscarinic and EP3-receptors, respectively in paratracheal ganglion cells. Parasympathetic nervous system also releases nonadrenergic-noncholinergic (NANC) inhibitory neurotransmitters in addition to ACh. The threshold level for ACh and NO release, for example, is almost identical, thereby suggesting the possoible interactions between NO and ACh at the post-junctional cites, in addition to the prejunctional inhibitory action of NO to suppress ACh release from the nerve terminal. In this context, NO-donors, including SNAP or Cys-NO, reduced the amplitude of carbachol-induced Ca2+-dependent Cl- currents (IOCh', ICl(Ca)) dose-dependently (IC50: about 10μM) in the cat tracheal myocytes, and similar inhibition was also exerted by dibutyryl cGMP (db-cGMP). However, two structually distinct types of G-kinase inhibitor, H-8 (2.5μM) and KT5823 (1μM) failed to counteract the inhibitory effects of SNAP or db-cGMP. Another G-kinase-specific inhibitor, (Rp)-8-pCPT-cGMPS, itself caused a marked reduction in IOCh' thereby indicating that inhibition of IOCh' by NO donors involves a cGMP-dependent but G-kinase-independent mechanism. All these experimental facts taken together indicate that there are pre and post-junctional “brakings” in the excitatory parasympathetic neuro-effector transmission. Furthermore, in the gastrointestinal tract, Z-338, a newly synthetized gastroprokinetic agent, stimulates gastrointestinal motility through inhibition of the pre-junctional braking system through inhibition of M1 receptors. Such a drug may be useful to control the respiratory and gastrointestinal smooth muscles.
  • 倉智 嘉久
    1998 年 112 巻 supplement 号 p. 32-35
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. They are widely distributed in various tissues including heart and vasculature. and thus may play essential regulatory roles in the cardiovascular system. Furthermore, KATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureaswhich block the channels, and a series of vasorelaxants called “K+ channel openers” which tend to maintain the channels in an open conformation. Recently, the molecular structure of KATP channels has been clarified in various tisseus including cardiovascualr system to be a complex of at least two subunits, i.e. SUR and Kir6.0. The KATP channels in heart and vascular smooth muscle now appear to be the complexs of SUR2A/Kir6.2 and SUR2B/Kir6.1, respectively. Further works are now in progress to understand the molecular mechanisms responsible for the control of KATP channel function by intracellualr nucleotides and drugs.
  • 井上 和秀, 上野 伸哉, 小泉 修一, 津田 誠
    1998 年 112 巻 supplement 号 p. 36-40
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    The possible involvement of adenosine 5'-triphosphate (ATP) receptors in the function of the hippocampus and of pain transmission is discussed. Involvement of these receptors in the function of the hippocampus has been suggested by several reports. In the paper we presented the data that ATP inhibits the glutamate release in cultured hippocampal neurons. This andthereport revealing that ATP protected against cell death by glutamatesuggest that ATP may be playing a role in the protection of the hippocampus from over-stimulation. Microglia cells are activatedby the stimulation of ATP andreleases plasminogen which is well known to promote the development of mesencephalic dopaminergic neurons and enhance neurite outgrowth from explants of neocortical tissue. Therefore, ATP may have a role in repairing the damaged neuronal networks as well as protection.
  • 吉井 光信, 西崎 知之, 渡部 繁男
    1998 年 112 巻 supplement 号 p. 41-43
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Nootropics are proposed to serve as cognition enhancers. The underlying mechanism, however, is largely unknown. We have attempted to assess the intracellular signal transduction pathways mediating the action of nefiracetam, a nootropic agent, on neruronal Ca2+ channels and nicotinic ACh receptors. In NG108-15 cells, nefiracetam (1 μM) enhanced the activities of N/L-type Ca2+ channels without affecting T-type. The nefiracetam action was mimicked by dibutyryl cAMP (1 mM), or blocked by pertussis toxin (PTX), indicating that PTX-sensitive inhibitory G-proteins and cAMP-dependent pathways mediate the drug action. Nefiracetam also exerted a dose-dependent biphasic effect on Torpedo nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes, in which the drug induced a short-term depression of ACh-evoked currents at submicromolar concentrations (0.01 - 0.1 μM) and a long-term enhancement of the currents at micromolar concentrations (1 - 10 μM). The depression was caused by activation of PTX-sensitive G-protein-regulated cAMP-dependent protein kinase (PKA) with subsequent phosphorylation of the ACh receptors; in contrast, the enhancement was caused by activation of Ca2+-dependent protein kinase C (PKC) and the ensuing PKC phosphorylation of the receptors. It is concluded that nefiracetam interacts with PKA and PKC pathways, which may explain a cellular mechanism for the action of cognitive enhancers.
  • 赤池 紀扶, 大村 和広
    1998 年 112 巻 supplement 号 p. 44-47
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Effects of arginine vasopressin (AVP(1-9)) and its analogues on the glycine (Gly)-induced Cl- currents (IGly) were examined in acutely dissociated rat hippocampal CA1 neurons using nystatin perforated and conventional whole-cell patch recording modes under voltage-clamp conditions. The results suggest that the activation of V1 receptor induces IP3 production which release Ca2+ from IP3-sensitive Ca2+ storage sites. The Ca2+ binds to CaM, resulting in theactivation of Ca2+/CaM-sensitive adenylate cyclases. Consequently, the activation of PKA inhibits IGly. The inhibitory actions of NC-1900 and its analogues on IGly might be involved in their ameliorating effects on impairments of learning and memory in. the CA1 neurons.
  • 谷内 一彦, 渡邉 建彦, 岡村 信行, 伊藤 正敏
    1998 年 112 巻 supplement 号 p. 48-52
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Antihistamines are the efficacious drugs to be used for the symptomatic relief of allergic diseases. The safety issue of antihistaminesis of central importanceb ecause of their widespreaduse in current medical practice. Positrone mission tomography (PET) was used to better understand the pharmacological effects of antihistamines on the central nervous system. The H1 receptor occupancy was examined in young male volunteers with [11C]-doxepianf ter the oral or intravenous administration of antihistamines. In other studies, the cognitive performance was also measured tachistoscopically before and after taking antihistamines. The H1 receptor occupancy in the human cortex caused by antihistaminesis significantly correlated with the reported values of incidence of sleepiness in clinical trials, and the occupancy is wellproportional to the impaired cognitive performance. To understand the brain mechanism of antihistamine-induced “sleepiness and impaired cognition”, the regional cerebral blood flow (rCBF) during the task was measured using 3D-PET and H215O before and after administration of d-chlorpheniramine. After its administration, the rCBF was significantly decreased on the bilateral middle temporal gyrus, midbrain and anterior cingulate. These findings suggest that H1 receptor blockade would be affected on the activity of the attention and cognitive system in the brain.
  • 柴田 和彦, 牧野 郁子, 芝口 浩智, 丹羽 正美, 大神 祐輔, 藤原 道弘, 古川 達雄, 桂木 猛
    1998 年 112 巻 supplement 号 p. 53-57
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    The present study examined changes in angiotensin type-2(AT2) receptor mRNA level after global brain ischemia or during glutamate neurotoxicity in cultured cortical cells in rats. The AT2 mRNA level increased by three-fold in both the cortex and hippocampus, which are known to be sensitive to ischemic injury, 3 hr after ischemia. The day 10-14 cortical neurons were exposed to glutamate at a toxic concentration of 100 μM for 15 min. AT2 receptor mRNA was then increased 2-fold after exposure to glutamate, while the maximum increase was observed in a dose-dependent manner 3 hr after glutamate stimulation. AT2 receptor binding also increased 3-12 hr after glutamate exposure. The increase in the mRNA level was antagonized by N-nitro L-arginine methyl-ester, a nitric oxide synthase inhibitor. The hemoglobin, a nitric oxide trap, also inhibited the increase in the mRNA level. These results suggest that the increase in the mRNA level is associated with the nitric oxide synthesis by glutamate exposure. The viability of cortical cells after glutamate stimulation was partially restored by the antisense oligonucleotide for the AT2 receptor. The present results thus suggest the AT2 receptor may in some way be related to one of the processes in cell injury.
  • 岡部 栄逸朗
    1998 年 112 巻 supplement 号 p. 58-62
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Because the net Ca2+ uptake in the sarcoplasmic reticulum (SR) of cardiac muscle is a result of the activity of Ca2+-ATPase and of the SR Ca2+-release channel, an abnormal Ca2+ uptake may be the result of the dysfunction of either or both structures. The site or sites of action for oxygen-derived free radicals (OFR) damage are unknow, although previous studies on the SR have focused on damage to the Ca2+ pump. Direct effects of OFR on SR Ca2+-release channels may be important in understanding their potential contribution to myocardial ischemia/reperfusion injury. We confirmed that superoxide anion radical (O2·-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles. Electron spin resonance study showed that hydroxyl radicals are generated in addition to O2·- from hypoxanthine-xanthine oxidase reaction, and data indicate that O2·- is responsibe for the observed effect. Current fluctuations through single Ca2+-release channels have been also monitored after incorporation into planar phospholipid bilayers. We directly demonstrate that activation of the channel by O2·- stimulates Ca2+release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2·-.
  • 尾崎 博, 山脇 英之, 佐藤 晃一, 堀 正敏, 唐木 英明
    1998 年 112 巻 supplement 号 p. 63-67
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    In the endothelium-denuded arteries cultured in the presence of FBS, morphological (i.e. smooth muscle disorientation and increase in collagen fiber) and phenotypic changes in smooth muscle were observed. Correlated with these changes, contractile force induced by high concentration of KCl and norepinephrine was significantly decreased. In addition, Ca-induced contraction in the permeabilized muscle was also significantly reduced. The reduced contractility in the FBS-treated arteries was partially recovered by the treatment with L-NMMA. In the endothelium-intact arteries cultured in the presence of FBS or PDGF, substance P and ionomycin-mediated, endothelium-dependent relaxation (EDR) was significantly decreased compared to the arteries cultured in serum-free condition. In addition, amounts of NO production and total recoverable eNOS mRNA was reduced in the FBS and PDGF-treated arteries. In these arteries, however, cGMP-dependent relaxation in smooth muscle was not impaired. These results suggest that long-term treatment of vascular tissue with growth-activating agents causes morphological or phenotypic changes and up-regulation of NO production in smooth muscle, resulting in a reduced contractility. Furthermore, long term treatment with these agents impairs NO-mediated EDR by decreasing eNOS mRNA and NO production.
  • 黒瀬 等, 瀧川 恵美子, 赤羽 悟美, 田中 里依, 長尾 拓
    1998 年 112 巻 supplement 号 p. 68-72
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    G protein-coupled receptor kinases (GRKs) are believed to involve in desensitization of the G protein-coupled receptors. So far, cDNAs of six GRKs were cloned from several species including human and rat. However, it is unknown whether single GRK phosphorylates various receptors and desensitizes them in the cells. To determine whether GRK2 (also called βARK1) involves desensitization of the β1-adrenergic receptor-mediated response in heart, we tried to apply monoclonal antibody which could recognize only βARK1 and inhibit its phosphorylating activity to the heart cells. Monoclonal antibody was obtained by immunization of carboxyl terminus of βARK1 as fusion protein of glutathione-S-transferase (GST). The resulting monoclonal antibody specifically reacted with βARK1, and inhibited the binding of purified βγ subunit to the carboxyl terminus. Monoclonal antibody completely inhibited phosphorylation of the m2 muscarinic acetylcholine receptor as well as phosphorylation of GST intracellular third loop fusion protein of the m2 receptor. When monoclonal antibody was applied to myocyte prepared from guinea pig heart, the desensitization of the β1-adrenergic receptor was partially inhibited as measured by Ca2+ channel activation. Thus intracellular application of monoclonal antibody is promising approach to analyze function of GRKs.
  • 赤木 正明, 福石 信之, 阪口 政清, 松井 敦聡, 高橋 秀明
    1998 年 112 巻 supplement 号 p. 73-77
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Rat peritoneal mast cells are stimulated to generate superxide anion (O2-) by the addition of compound 48/80 and A23187. Recently, we demonstrated by immunohistochemical and Western blot analysis that the mast cells contained the p47phox protein, which was one of cytosolic component of the NADPH oxidase system. In the present study, it was demonstrated that the mast cells contained the p47phox mRNA, much similar to that of mouse leukocyte. The permeabilized mast cells were stimulated to generate O2- by the addition of Ca2+, phospholipase A2(PLA2) and arachidonic acid. Our data suggest the following: (1) cytosolic PLA2 may be activated by the elevation of [Ca2+]i; (2) the conjugation of membrane component with cytosolic component may be stimulated by the released arachidonic acid. The mast cell granules contained superoxide dismutase (SOD)-like enzyme, which degradated O2-, generated in xanthine-xanthinoxidase system. SOD-like enzyme was released from the granules by the treatment with Ca2+ and trapped by the treatment with heparin. In conclusion, our studies suggest that the disorder of the degradation system of O2- may contribute to the development of allergic inflammation.
  • 中川 貴之, 小澤 徹, 渡辺 豪, 南 雅文, 佐藤 公道
    1998 年 112 巻 supplement 号 p. 78-82
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Chronic opioid treatment has been shown to develop supersensitization of adenylyl cyclase (AC) system or cAMP overshoot. In this study, we investigated the molecular mechanism of supersensitization of AC system using CHO cells expressing one of the cloned μ-, δ- and κ-opioid receptors. In naive cells, acute treatment with an opioid agonist, but not antagonist, suppressed forskolin-stimulated cAMP accumulation. In the cells sustainedly (4 hr) treated with the agonist, the challenge by antagonist induced the cAMP overshoot over the naive level (supersensitization of AC system), but had no effect on GTPase activity. This supersensitization of AC system was not affected by pretreatment with cycloheximide, a protein synthesis inhibitor, or various protein kinase inhibitors (H7, H8, H89 and staurosporine). On the other hand, pretreatment with pertussis toxin blocked both inhibition of AC activity by acute agonist treatment and development of supersensitization of AC system. To examine an involvement of the interaction between G protein and AC in the supersensitization of AC system, we used CHO cells coexpressing the opioid receptor and some chimeric Ga proteins between Gαi2 and Gαq. The results revealed that a specific region of Gαi2, which is responsible for the interaction with AC, is closely related to the supersensitization. In addition, the supersensitization of AC system was induced by sustained muscarinic agonist treatment in CHO cells expressing the cloned m2 or m4 muscarinic receptor, suggesting this phenomenon is common to the members of the Gi-coupled receptor superfamily. In conclusion, these findings suggest that the development of supersensitization of AC system is attributed to a continuous inhibition of AC by Gαi, but not to continuous activations of the Gi-coupled receptor and G protein themselves.
  • 森本 潔, 長田 聖子, 久保 武一, 尾田 富一郎, 金子 勲
    1998 年 112 巻 supplement 号 p. 83-87
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    We have previously shown that in vivo injection of β-amyloid (β1-40, β25-35) with non-toxic amounts of ibotenic acid, an excitatory amino acid, causes synergistic and drastic neuronal degeneration in rat hippocampus. It was, however, yet not clear whether the neuronal degeneration in vivo was associated with their primary amino acid sequences, their secondary β-structureor their activities to suppress MTT reduction activity in vitro. In addition to β-amyloid, other amyloidogenic peptides such as human amylin or calcitonin are known to deposit extracellularly in systemic and peripheral amyloidosis. In this study, we measured the activity of amyloidogenic peptides (β1-40, human amylin and calcitonin) to suppress cellular MTT reduction activity in vitro and their synergistic neurodegeneration with ibotenic acid in vivo. All amyloidogenic peptides, but not non-amyloidogenic peptides (β40-1, BSA), suppressed the MTT reduction activity in HeLa cells and in the primary cultured neurons in vitro, and also produced the synergistic neuronal cell loss in rat hippocampal region by enhancing the toxicity of ibotenic acid in vivo. The deposits of amyloidogenic peptides at the injection sites were thioflavin S fluorescence positive, suggesting the fibrillary β-structures. These results indicate that the neurodegeneration in vivo by amyloidogenic peptides is strongly associated with their fibrillary β-structurea nd their activity to suppress MTT reduction activity in vitro.
  • 岩崎 克典, 鄭 恩希, 藤原 道弘
    1998 年 112 巻 supplement 号 p. 88-92
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    We have been reported that the repeated cerebral ischemia induced more severe disruption of spatial cognition than single ischemia without any other motor disturbance in 8-arm radial maze task in rats. And we have been clarified that it is corresponding with 60% of selective cell injury of the CA1 pyramidal cells in the hippocampus. Recently, characteristics of apoptosis such as internucleosomal DNA fragmentation have been found in excitotoxic neuronal death. In the present study, we investigated how necrosis and apoptosis following repeated ischemia involve to the cell death. Repeated cerebral ischemia (10 min × 2, 1hr interval) induced significant disruption of spatial cognition not only 24hrs but also 7 days after reperfusion. The decrease of H.E-positive neurons was found in the hippocampus CA1 area and frontal cortex within 3 days after reperfusion, while an DNA fragmentation and TUN-NEL-positive neurons in the same areas were found afterward. Furthermore repeated cerebral ischemia-induced disruption of spatial cognition and apoptosis in the hippocampal CA1 area were inhibited by YM-90K(15mg/kg, i.p.), which is a selective AMPA/KA receptor antagonist, but not by MK-801.These results suggested that the apoptotic cell death may be occured via non-NMDA receptor mechanism in relatively late phase of the reperfusion period and it may relate to the incidence of the disruption of spatial cognition in the rat.
  • 三須 良實, 古川 信也, 新井 信隆, 宮前 丈明, 五嶋 良郎
    1998 年 112 巻 supplement 号 p. 93-97
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    DOPA itself is a neuromodulator in striata. In rat striata, DOPA by itself released neuronal glutamate in slices and caused cell death via glutamate release in cultured fetal neurons, suggesting involvement of DOPA in an upstream process of mechanisms for in vivo neuronal cell death. We attempted to clarify whether or not this idea is truly the case in conscious Wistar rats. Four vessels were occluded for 10 min during microdialysis of striata. DOPA, dopamine and glutamate in perfusates collected every 10 min were measured by HPLC-ECD and spectrophotometer. Delayed neuronal cell death in striata and hippocampus was evaluated 96 hr after ischemia. DOPA was indeed evoked with dopamine and glutamate during and after ischemia, and peak increases by respective 6-, 210- and 8-fold of a basal level were seen at the fraction immediately after ischemia. Delayed neuronal cell death was slight to moderate in striata and severe in hippocampus. Intrastriatal perfusion of NSD-1015, a central DOPA decarboxylase inhibitor, at30 μM 10 min before ischemia, markedly increased DOPA and glutamate release by ischemia with slight inhibition of dopamine release and exaggerated delayed neuronal cell death in striata. Meanwhile, intrastriatal perfusion of DOPA cyclohexyl ester (DOPA CHE) at 10-100 nM, a novel stable potent competitive DOPA antagonist, antagonized dosedependently increases in glutamate release by ischemia without modification of dopamine release. DOPA CHE at 100 nM protected striatal neurons from delayed cell death. Hippocampal neuronal cell death was neither affected by NSD-1015 nor by DOPA CHE. Endogenously released DOPA itself seems to act on its recognition site and to be a causal factor for increase in glutamate release and resultant delayed neuronal cell death by transient ischemia in rats.
  • 久米 利明, 香月 博志, 金子 周司, 赤池 昭紀
    1998 年 112 巻 supplement 号 p. 98-102
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    This study was performed to investigate the effects of neurotrophins on glutamate cytotoxicity by using cultured cortical neurons. Primary cultures obtained from the cerebral cortex of fetal rats (17-19 days gestation) were used for experiments. NGF did not elicit tyrosine phosphorylation of Trks whereas BDNF induced Trk tyrosine phosphorylation within 10 min, followed by time-dependent decrease. Brief glutamate exposure to the cell induced delayed cytotoxicity. Similar cytotoxicity was observed with the brief application of a calcium ionophore, ionomycin, and nitric oxide (NO) generating agents, S-nitrosocysteine (SNOC) and SIN-1. Exposure of the cultures to NGF and BDNF for 1 or 24 hr prior to glutamate exposure reduced glutamate-induced cytotoxicity. In contrast, simultaneous addition of NGF and BDNF with glutamate did not affect glutamate-induced cytotoxicity. Ionomycin-induced cytotoxicity was prevented by exposing cultures to NGF and BDNF for 24-hr. Moreover, NGF and BDNF ameliorated cytotoxicity induced by SNOC and SIN-1. These results suggest that neurotrophins prevent NO mediated glutamate cytotoxicity.
  • 立石 成人, 鏡石 佳史, 新宅 克也, 下田 泰治, 品川 理佳, 佐藤 壮一, 近藤 規元
    1998 年 112 巻 supplement 号 p. 103-107
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Astrocytes play vital roles not only in the mechanical support of the central nervous system but also in the metabolism of neurotransmitters and in the transfer of nutritive substances to neuron. After ischemic brain injuries, it has been known that gliosis appears around degenerative regions and repairs these regions. Recently, accumulating evidence indicates that overexpression of S-100 protein, astrocyte-derived protein, is detrimental to neuronal cells in various pathological conditions. To confirm the astrocytic activation in cerebral ischemia, we examined immunohistochemical changes in S-100 protein and glial fibrillary acidic protein (GFAP) in the transient focal ischemia. Cerebral infarction determined by hematoxylin-eosin staining was slight on day 1 and further expanded on day 2 and 3. Thereafter, GFAP immunoreactivity was observed in boundary zone of the infarct area at 72 hours after the transient focal ischemia. On the other hand, S-100 protein immunoreactivities were markedly increased at 9 hours after the transient focal ischemia. After the infarct formation, the increase of S-100 immunoreactivity was observed in outside boundary of infarct area. These results suggest that astrocytic activation, which we would like to be called “pre-mitotic S-100 peak (PSP)”, precedes the neurodegeneration following the transient focal ischemia, and should be distinguished from so-called gliosis observed in the post-neurodegeneration and GFAP-dependent astrocytic proliferation.
  • 田中 康一, 大西 佳子, 佐藤 友昭, 西川 殷維
    1998 年 112 巻 supplement 号 p. 108-112
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    In the CNS, reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including amyotrophic lateral sclerosis, ischemia-reperfusion injury, Alzheimer disease, Parkinson disease and aging. However, the exact mechanism is unknown, and there is little information on possible roles of ROS in cell injury and the process on recovery of astrocytes, the most abundant glial cells in the brain. We examined hydrogen peroxide (H2O2)-inducedDNA fragmentation and thymidine incorporation into cultured astrocytes as an indicator of the process of recovery from astrocytic DNA injury. Astrocytes were isolated from cerebral cortices of 0-day-old rats and treated with 1 mM dibutyryl cyclic AMP for 4 days. H2O2 of 100 μM stimulated thymidine incorporation into astrocytes. Caffeine, ryanodine, cyclic ADP-ribose (endogenous ryanodine receptor agonist) and β-NAD+ (precursor of cyclic ADP-ribose) suppressed partially the stimulatory effect of H2O2. Ruthenium red (ryanodine receptor antagonist) facilitated further the stimulatory effect of H2O2. The facilitated effect of ruthenium red on H2O2-induced thymidine incorporation was suppressed by caffeine, ryanodine, cyclic ADP-ribose and β-NAD+. H2O2-induced DNA fragmentation and astrocytic death were suppressed by ruthenium red. These findings suggest that the process of recovery from astrocytic DNA injury by H2O2 may be regulated by Ca2+ efflux from ryanodine-sensitive intracellular Ca2+ stores.
  • 中畑 則道, 本間 茂佳, 小林 博, 大泉 康
    1998 年 112 巻 supplement 号 p. 113-117
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Thromboxane A2 (TXA2) receptor subtypes and their signal transduction were examined in 1321N1 human astrocytoma cells. Placental and endothelial types of TX2 receptor mRNA were found in astrocytoma cells by RT-PCR procedure. Using immunoaffinity column conjugated with anti-TXA2 receptor antibody, two TXA2 receptors (58 and 55 kDa) were partially purified. The partially purified TXA2 receptor fraction contained Gq/11 and G12. The incubation of the cells with dibutyryl cyclic AMP (dbcAMP) caused a differentiation of human astrocytoma cells. DbcAMP treatment resulted in the reduction of Ca2+ elevation and phosphoinositide hydrolysis induced by TXA2 receptor agonist. Whereas the responsiveness of Ca2+ signaling was weakened by dbcAMP treatment, phosphorylation of mitogen-activated protein kinase was increased in dbcAMP-treated cells. These results suggest that human astrocytoma cells express placental and endothelial TXA2 receptors. The TXA2 receptors were coupled with Gq/11 and G12. DbcAMP treatment discriminates TXA2 receptor-mediated MAPK activation from the Ca2+ signaling pathway.
  • 上原 孝, 野村 靖幸
    1998 年 112 巻 supplement 号 p. 118-122
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    We here report involvement of caspases in NO-induced neuronal apoptosis. Our experiments were designed to elucidate how NO induces neuronal cell death using NOC18, a new type of NO donor that spontaneously releases NO alone, without enzymatic metabolization. NOC18 induced apoptosis in human neuroblastoma SH-SY5Y cells in a concentration- and time-dependent manner estimated with DNA fragmentation assay, FACScan analysis, and nuclear morphology. In this study, oxyhemoglobin, an NO trapper, suppressed NOC18-triggered DNA fragmentation, indicating that NO from NOC18 is an apoptosis-inducer. An increase in caspase-3-like protease activity was observed in parallel with the induction of apoptosis, but no caspase-1-like protease activity was detected. The level of pro-caspase-2 protein, a precursor of caspase-2, was decreased dramatically. In addition, NOC18 also caused the cleavage of PARP, yielding an 85 kDa protein, a typical fragment of the caspases reaction. Oxyhemoglobin blocked the decrease in pro-caspase-2 and the cleavage of PARP by NOC18. Moreover, NO elicited the release of cytochrome c into the cytosol from mitochondria during apoptosis. These results suggest that activation of caspases by cytochrome c released from mitochondria is involved in neuronal apoptosis induced by NO.
  • 矢富 義信, 原 周司, 福澤 美佐, 小野 信文, 黒田 健
    1998 年 112 巻 supplement 号 p. 123-127
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    We are investigating the influence of NO synthetase inhibitor on the clonidine-induced cardiovascular actions in urethane-anesthetized rats. The systemic blood pressure was measured from right femoral artery, heart rate from the pressure pulse under inhalation of O2. Nw-nitro-L-arginine-methylester (L-NAME, 5 mg/kg), NO synthetase inhibitor, administered intravenously increased blood pressure slightly, although decreased heart rate. The responses to L-NAME were stable about 10 min after the injection. Clonidine (5 mg/kg) administered intravenously indicated the transient increase blood pressure and following continuous decrease of blood pressure. The early transient hypertension of clonidine was potentiated by pretreatment with L-NAME and later continuous hypotension was markedly inhibited. While, the early transient hypertension of clonidine was inhibited by pretreatment with L-arginine and later continuous hypotension was potentiated. The hypertension and tachycardia of intravenous tyramine was enhanced by L-NAME. Clonidine administered into the cerebroventricle did not indicate the early transient hypertension, though produced the later continuous hypotension. These results suggest that L-NAME modifies the cardiovascular responses to clonidine and NO may participate in the modulation.
  • 木村 郁子, 野島 浩史, M.A. ISLAM
    1998 年 112 巻 supplement 号 p. 128-132
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    We investigated the involvement of muscarinic M1 receptors in the regulation of action potentials, and its modulation by adrenergic signaling and its change by aging in mouse isolated right atria using a conventional glass microelectrode technique. In adult mice, acetylcholine (ACh) (3-10 μM) reduced the maximum upstroke velocity of action potential (Vmax) followed by an increase. In electrically driven atria, similar effects of ACh on Vmax were observed. McN-A-343 (100-300 μM), a M1 agonist, reduced Vmax, while M2 agonist oxotremorine (0.1-0.3 μM), increased it. Isoproterenol (3 nM), antagonized ACh- and McN-A-343-induced reduction of Vmax, and potentiated the ACh- and oxotremorine-induced increase. The effects of isoproterenol were mimicked by cholera toxin, a Gs-protein activator, and forskolin, a direct activator of adenylyl cyclase. H-89, a selective protein kinase-A inhibitor, abolished the antagonism by isoproterenol of ACh-induced reduction in Vmax. Calphostin C, a selective protein kinase-C inhibitor, but not pertussis toxin attenuated ACh-induced reduction in Vmax. These results show that 1) ACh-induced reduction of Vmax and its subsequent increase are mediated by the activation of muscarinic M1 and M2 receptors, respectively, 2) the M1 and M2 subtypes may exert a balancing action on each other, and 3) the β-adrenergic activation antagonizes M1-mediated effects, and enhances M2-mediated effects, on Vmax. In young mice, ACh (5-10 μM) increased Vmax, which was abolished by AF-DX 116 (0.3 μM), a M2 antagonist. In aged mice, ACh did not affect Vmax up to a concentration of 10 μM. The present findings may be of importance in the occurrence of cardiac disfunction in aging.
  • 平藤 雅彦, 加藤 健治, 鈴口 智子, 小川 隆志, 遠藤 泰, 佐藤 洋一, 南 勝
    1998 年 112 巻 supplement 号 p. 133-137
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Although serotonin (5-HT) release from enterochromaffin (EC) cells is considered to be regulated by multiple receptor-mediated mechanisms, little is known about the signal transduction in EC cells. We investigated the effects of adrenoceptor stimulation on 5-HT release from ileal tissue and intracellular calcium dynamics of epithelial cells in isolated ileal crypts in mice. Ileal tissues placed in organ bath were perfused with a buffered solution. Released 5-HT was measured using HPLC-ECD. Ileal crypts were isolated by collagenase digestion followed by moderate pipetting. Intracellular calcium dynamics were analyzed by digital video-imaging system using fura-2. NE, but not isoprenaline (Iso), induced 5-HT release from mouse ileal tissue. NE-induced 5-HT release was antagonized by yohimbine and rauwolscine, but not by prazosin and bunazosin. NE, but not Iso, also elicited a transient elevation of intracellular calcium in some EC cells. The effect of NE (1 μM) was slightly suppressed by prazosin and bunazosin, but was remarkably suppressed by yohimbine and rauwolscine. UK 14, 304 and Clonidine at 10 μM significantly induced an increase in intracellular calcium concentration. NE-induced intracellular calcium dynamics was not significantly affected by timolol, Ro20-1724, rolipram and 8-bromo-cAMP. These results suggest that NE-induced 5-HT release from ileal EC cells is mediated predominantly via α2-, but not β-adrenoceptors, by a mechanism dependent on elevation of intracellular calcium concentration.
  • 原 英彰, 嶋澤 雅光, 橋本 宗弘, 洲加本 孝幸
    1998 年 112 巻 supplement 号 p. 138-142
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    Lomerizine, a novel Ca2+ channel blocker, is under development as an anti-migraine drug. We examined the effects on spreading depression (SD) induced by a brief period of hypoxia (40 to 60 sec) in rat hippocampal slices, the cortical hypoperfusion and cortical c-Fos-like immunoreactivity that follow KCl-induced SD in anesthetized rats as compared with those of flunarizine. Extracellular recording was made from the CAl subfield. The latency of initiated SD was examined. Lomerizine (1 and 10 nM) and flunarizine (1 μM) significantly prolonged the latency in a concentration-dependent manner. After KCl application to the cortex, cerebral blood flow monitored by the laser Doppler flowmetry was approximately 20 to 30% below baseline for at least 60 min. Lomerizine (0.3 and 1 mg/kg, i.v.) and flunarizine (1 and 3 mg/kg, i.v.) administered 5 min before KCl application inhibited the cortical hypoperfusion that followed KCl application. c-Fos-like immunoreactivity, an indicator of neuronal activation, was detected in the ipsilateral, but not in the contralateral frontoparietal cortex 2 hr after KCl application. Lomerizine (3-30 mg/kg, p.o.) and flunarizine (30 mg/kg, p.o.) significantly attenuated the expression of c-Fos-like immunoreactivity in the ipsilateral frontoparietal cortex. Lomerizine was 3 to 1000 times more potent than flunarizine in the above SD models. These findings suggest that the inhibitory effects of lomerizine and flunarizine on the interval between the initiated and subsequent spontaneous SDs, the cortical hypoperfsuion and expression of c-Fos-like immunoreactivity induced by SD are mediated via the effects of Ca2+ entry blockade, which may include an increase in cerebral blood flow and the prevention of excessive Ca2+ influx into brain cells.
  • 藤本 勝巳, 大田 和美, 寒川 賢治, 吉川 潮, 荻野 敏, 福井 裕行
    1998 年 112 巻 supplement 号 p. 143-147
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    ヒスタミンH1受容体情報伝達は蛋白キナーゼCを活性化するホルボールエステルを与えることにより脱感作される。クローニングにより構造決定されたヒトH1受容体の第3細胞内領域には蛋白キナーゼCのリン酸化可能部位としてのコンセンサス配列が相当数見いだされた。そこで、蛋白キナーゼCによるH1受容体の直接のリン酸化が脱感作を引き起こすかどうかを調べた。ヒトH1受容体の第3細胞内領域のセリン-396およびセリン-398が蛋白キナーゼCのリン酸化部位と考えられた。そして、セリン-398のリン酸化がH1受容体の脱感作に大きく関与することが明らかとなった。
  • 右田 啓介, 堀 信顕, 斎藤 亮, 本多 健治, 高野 行夫, 山本 健二, 神谷 大雄
    1998 年 112 巻 supplement 号 p. 148-152
    発行日: 1998年
    公開日: 2007/01/30
    ジャーナル フリー
    The effects of vasopressin (AVP) on area postrema (AP) neurons and the neuronal connection between the AP and nucleus tractus solitarii (NTS) were investigated electrophysiologically in slices perparation of the medulla oblongata of rats. In the AP, 27.9% of 129 neurons were excited by AVP and 20.5% were inhibited. The excitation was blocked by a V1 receptor antagonist. Synaptic transmission of the AP to the NTS was mainly mediated by non-NMDA receptors. Local application of AVP to the AP activitied the NTS neurons. This activation was blocked by an NMDA antagonist. These results suggest that the excitation originating in the AP is conveyed to the NTS via non-NMDA receptors and modified by NMDA receptor activation secondly. These processes may be improtant in regulation of the arterial baroreceptor reflex.
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