yoichi nakamura - Academia.edu (original) (raw)
Papers by yoichi nakamura
Biological & Pharmaceutical Bulletin, 2002
Microglia, residential macrophages in the central nervous system, can release a variety of factor... more Microglia, residential macrophages in the central nervous system, can release a variety of factors including cytokines, chemokines, etc. to regulate the communication among neuronal and other types of glial cells. Microglia play immunological roles in mechanisms underlying the phagocytosis of invading microorganisms and removal of dead or damaged cells. When microglia are hyperactivated due to a certain pathological imbalance, they may cause neuronal degeneration. Pathological activation of microglia has been reported in a wide range of conditions such as cerebral ischemia, Alzheimer's disease, prion diseases, multiple sclerosis, AIDS dementia, and others. Nearly 5000 papers on microglia can be retrieved on the Web site PubMed at present (November 2001) and half of them were published within the past 5 years. Although it is not possible to read each paper in detail, as many factors as possible affecting microglial functions in in vitro culture systems are presented in this review. The factors are separated into "activators" and "inhibitors," although it is difficult to classify many of them. An overview on these factors may help in the development of a new strategy for the treatment of various neurodegenerative diseases.
Neuroscience Research Supplements
Tight-seal whole-cell clamp experiments were performed on thin slices (150-250 p) obtained from t... more Tight-seal whole-cell clamp experiments were performed on thin slices (150-250 p) obtained from the caudate nucleus of rats (9-14 days old). Most of the small cells (lo-15 w in diameter) stained with Lucifer Yellow exhibited long dendrites stubbed with fine spines, which is characteristic of medium-sized spiny neurons. Focal stimulation of the caudate nucleus with a glass pipette purely evoked excitatory synaptic currents in the presence of picrotoxin. The EPSCs were composed of NMDA and non-NMDA components; APV abolished the former component and CNQX the latter. Spontaneous EPSCs also comprised the two components, suggesting co-localization of the two receptor subtypes in the postsynaptic membranes. One feature of the ghttamatergic synaptic transmission was that, in the paired-pulsed experiments, the second EPSC was inhibited to 30% of the first EPSCs. This marked paired-pulse inhibition occurred both in the NMDA and non-NMDA components, showed no delay in onset and persisted up to 500 ms in the NMDA components, but only to 30 ms in the non-NMDA component. Quantal analysis suggested the involvement of a presynaptic mechanism in the inhibition of the non-NMDA component.
Neurochemistry International
Microglial activation has been suggested to play important roles in various neurodegenerative dis... more Microglial activation has been suggested to play important roles in various neurodegenerative diseases by phagocytosis and producing various factors such as nitric oxide (NO), proinflammatory cytokines. Excessive production of NO, as a consequence of increased inducible nitric oxide synthase (iNOS) in microglia, contributes to the neurodegeneration. During a search for compounds that regulate endoplasmic reticulum (ER) stress, a dibenzoylmethane derivative, 2,2'-dimethoxydibenzoylmethane (DBM 14-26) was identified as a novel neuroprotective agent (Takano et al., Am. J. Physiol. Cell Physiol. 293, C1884-1894, 2007). We previously reported in cultured astrocytes that DBM 14-26 protected hydrogen peroxide-induced cell death and inhibited lipopolysaccharide (LPS)-induced NO production (Takano et al., J. Neurosci. Res. 89, 955-965, 2011). In the present study, we assessed the effects of DBM 14-26 on microglia using the mouse cell line BV-2 and found that DBM 14-26 inhibited LPS-induced iNOS expression and NO production also in microglia. DBM 14-26 also suppressed LPS-induced IL-1β expression. Conditioned medium of BV-2 cells stimulated by LPS significantly decreased cell viability of neuron (human neuroblastoma SH-SY5Y cells) compared with the absence of LPS. Conditioned medium of BV-2 cells stimulated by LPS in the presence of DBM 14-26 did not significantly decreased cell viability of neuron. These results indicate that microglial activation by LPS causes neuronal cell death and DBM 14-26 protect neuron through the inhibition of microglial activation. Functional regulation of microglia by DBM 14-26 could be a therapeutic candidate for the treatment of neurodegenerative diseases.
Neurochem Res, 1994
From rat hippocampal homogenate, we recently isolated a novel subcellular fraction richly contain... more From rat hippocampal homogenate, we recently isolated a novel subcellular fraction richly containing glial plasmalemmal vesicles (GPV), which takes up glutamate remarkably as a synaptosomal fraction [Y. Nakamura et al. (1993) Glia, 9, 48-56]. In the present study, we prepared GPV from different regions of rat CNS, namely olfactory bulb (Ob), cerebral cortex (Cx), caudatoputamen (Cp), hippocampus (Hp), cerebellum (Ce) and spinal cord (Sc), and analyzed their activities of Na+-dependent uptake of following neurotransmitters and a related compound; glutamate, ",/-aminobutyrate (GABA), glycine, dopamine and choline. The uptake activities of these amino acids were not significantly different between GPV and synaptosomes in each region. Regionally, however, the activities were varied considerably. The activities of glutamate uptake revealed in the following rank order: Cx, Hp, Cp > Ce, Ob > Sc. GABA uptake activities were: Ce > Ob, Cx, Hp > Cp, while glycine uptake activities were: Sc, Ce > Ob, Cp, Cx, Hp. On the other hand, the uptake activities of dopamine and choline were quite different between GPV and synaptosomes. Synaptosomal fraction from Cp took up dopamine in a high activity; however, GPV from the same tissue hardly showed the uptake activity. Choline was taken up by synaptosomes prepared from Hp but not by GPV.
J Neurochem, 2002
The time course of the decline in energy levels during an in vitro ischemia-like condition was co... more The time course of the decline in energy levels during an in vitro ischemia-like condition was compared with changes in intracellular Ca2+ concentration ([Ca2+]i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod-2. During the on-line monitoring of [Ca2+]i, the slices were perfused with an in vitro ischemia-like medium (33 degrees C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region-specific and acute [Ca2+]i elevations were observed in CA1 approximately 4 min after onset of the in vitro ischemia-like condition and also in the inner portion of the DG with a delay of 10-40 s. The change in ATP levels was related to the increase in [Ca2+]i. ATP levels in all subregions gradually decreased before the acute [Ca2+]i elevation. Concomitant with the acute [Ca2+]i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high-energy-charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca2+]i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region-specific increase in [Ca2+]i, which activates a Ca(2+)-ATP-driven pump and produces a subsequent fall in neuronal ATP content.
Journal of Biochemistry, Feb 1, 1978
Neurochem Res, 1995
In order to elucidate the mechanism of release of excitatory amino acid (EAA) induced by hypoxia-... more In order to elucidate the mechanism of release of excitatory amino acid (EAA) induced by hypoxia-hypoglycemia (in vitro ischemia) from cultured hippocampal astrocytes, we compared the EAA release by in vitro ischemia with those by other treatments. The EAA release induced by in vitro ischemia treatment was rapid and reversible. The amount of released aspartate was comparable to that of glutamate, although the endogenous content of aspartate was one sixth that of glutamate. High-K (100 mM) treatment and the addition of 5 mM NaCN induced a rapid EAA release and the glutamate release was much greater than aspartate. Addition of 5 mM iodoacetate, a glycolysis inhibitor, induced a slow EAA release, and the amount of released aspartate was much higher than that of glutamate. On the other hand, the in vitro ischemia treatment and the addition of 5 mM NaCN induced only 20% reduction in ATP content for initial 5 min, whereas the addition of 5 mM iodoacetate induced a marked reduction. Our data suggest that ischemia-induced EAA release from astrocytes is a complex process in which local energy failure, inhibition of glycolysis, and depolarization of the cell membrane are involved.
Endocrinology, Dec 1, 1986
Microsomal fractions prepared from porcine thyroid glands by differential centrifugations and suc... more Microsomal fractions prepared from porcine thyroid glands by differential centrifugations and sucrose density gradient centrifugation showed an ATP-dependent Ca2+ uptake. Electron microscopy and the study of marker enzyme activities suggested that the fractions consisted mainly of endoplasmic reticulum. The amount of transported Ca2+ increased four times in the presence of 20 mM oxalate owing to the precipitation of calcium oxalate, which was detected inside the microsomal vesicles by electron microscopy. Ca2+ was released rapidly when the calcium ionophore, A-23187, was added. The Ca2+ concentration for the half-maximal activation of Ca2+ transport was about 1 microM. These results indicate that Ca2+ is translocated into the lumen of microsomes against a concentration gradient in a manner of the active transport. The microsomes showed Ca2+-dependent ATPase activity and were phosphorylated by the reaction with [gamma-32P]ATP in a similar Ca2+ dependence to that of transport rate. A 105-kDa phosphoprotein was identified by dodecyl sulfate polyacrylamide gel electrophoresis and was found to be sensitive to hydroxylamine. These properties of the phosphoprotein were the same as those of Ca2+ pump ATPase in the endoplasmic reticulum of other cells. These results suggest that the cytosolic Ca2+ is maintained at low levels by the microsomal uptake of Ca2+ by the action of the ATP-dependent Ca2+ pump or active transport system.
Brain Res, 2003
Microglial proliferation and activation have been reported to occur after several central nervous... more Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10-300 microM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P(2)y receptor agonists (ADPbetaS and 2-methylthio-ATP) on LPS-induced TNF-alpha release. The rank order of inhibitory potency of endogenous purines on TNF-alpha release was: ATP>ADP>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 microM 2-methylthio-ATP showed a potency similar to 100 microM ATP. The addition of ADPbetaS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P(2)y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.
J Neurochem, 2008
Microglial activation has recently been recognized as a cause of damage in various neurodegenerat... more Microglial activation has recently been recognized as a cause of damage in various neurodegenerative diseases. A possible mechanism underlying this damage is the activation of microglia by serum factors leaked through a disruption of the blood-brain barrier, which in turn trigger microglial cell proliferation and the release of various substances toxic to neurons, such as superoxide (O(2)(-)). We recently reported that serum albumin enhanced O(2)(-) production in cultured rat microglia stimulated by phorbol ester. In the present report, we identify the active site of this enhancement within the albumin molecule. We purified an active subfragment from trypsin-treated bovine serum albumin that was composed of 12-mer and 33-mer peptides connected by a disulfide bond. The chemically synthesized 12-mer peptide showed activity within a concentration range ( approximately 10(-7) M:) equivalent to that of albumin. The activities of a series of synthesized peptides conclusively indicated that the minimum active sequence was Leu-His-Thr-Leu. The present study may shed light on the mechanism of neuronal cell damage in various neurodegenerative diseases.
Structure and Function of Sarcoplasmic Reticulum, 1985
NeuroImmune Biology, 2008
Neuroscience Research Supplements, 1994
Biochimica et biophysica acta, Jan 5, 2015
The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-pho... more The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusio...
Neuroimmunomodulation, Jan 3, 2014
Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be invol... more Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). Methods: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RT-PCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-µm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. Results: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulat...
The Journal of veterinary medical science / the Japanese Society of Veterinary Science, 2014
Amphotericin B (AmB) is a polyene antifungal drug and is reported to be one of a few reagents hav... more Amphotericin B (AmB) is a polyene antifungal drug and is reported to be one of a few reagents having therapeutic effects on prion diseases, that is, a delay in the appearance of clinical signs and prolongation of the survival time in an animal model. In prion diseases, glial cells have been suggested to play important roles; however, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotrophic factors in microglia and astrocytes (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290-1296; Motoyoshi-Yamashiro et al., 2013, ibid. 63, 93-100). These results suggested that neurotrophic factors derived from glial cells might be involved in the therapeutic mechanism of AmB. In the present study, we examined immunohistochemically the effects of AmB on the expression of neurotrophic factors in the rat brain. We found that direct injection of AmB into the striatum significantly enhanced the expression of glial cell...
Neurochemistry international, 2006
Uptake of K+ is an important role of astrocytes to maintain physiological lower extracellular K+ ... more Uptake of K+ is an important role of astrocytes to maintain physiological lower extracellular K+ concentration in the CNS. In this study, the effect of high K+ concentration was examined on the cellular function of astrocytes from embryonic rat brain in primary culture. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) was measured as an index of cellular function of astrocytes. Increasing KCl concentration to about 40 mM did not directly evoke NO production, but doubled the level of LPS (1 ng/ml)-induced NO production. K-gluconate showed a similar enhancing effect although the degree of enhancement was about half of that of KCl. Neither NaCl nor Na-gluconate showed any effect. The K(+)-channel blocker, 4-aminopyridine, but not tetraethylammonium or apamin, inhibited the enhancing effect of KCl. The LPS-induced iNOS protein expression determined by immunoblotting analysis was enhanced by high K+ treatment. The level of iNOS mRNA determined by real-time RT-PCR techniqu...
Current neurovascular research, 2005
The polyamines, putrescine, spermidine and spermine are present in most living cells, with the es... more The polyamines, putrescine, spermidine and spermine are present in most living cells, with the essentiality for normal cell function, cellular growth and differentiation. In the mammalian brain, polyamines are also present at relatively high concentrations with different regional distribution profiles. Cerebral ischemia is a leading cause of disability and mortality in humans, and believed to yield a cascade of cytotoxic molecules responsible for the death of viable cells in the brain. Polyamines have been implicated in the pathogenesis of ischemic brain damage. For example, polyamine biosynthesis is increased after the onset of cerebral ischemia through an induction of ornithine decarboxylase, a key enzyme in the polyamine biosynthetic pathway. The administration of a drug that inhibits ornithine decarboxylase activity prevents the development of ischemic brain damage, suggesting a critical role of the accumulation of polyamines in the ischemic brain in the pathogenesis of stroke. ...
Biological & Pharmaceutical Bulletin, 2002
Microglia, residential macrophages in the central nervous system, can release a variety of factor... more Microglia, residential macrophages in the central nervous system, can release a variety of factors including cytokines, chemokines, etc. to regulate the communication among neuronal and other types of glial cells. Microglia play immunological roles in mechanisms underlying the phagocytosis of invading microorganisms and removal of dead or damaged cells. When microglia are hyperactivated due to a certain pathological imbalance, they may cause neuronal degeneration. Pathological activation of microglia has been reported in a wide range of conditions such as cerebral ischemia, Alzheimer's disease, prion diseases, multiple sclerosis, AIDS dementia, and others. Nearly 5000 papers on microglia can be retrieved on the Web site PubMed at present (November 2001) and half of them were published within the past 5 years. Although it is not possible to read each paper in detail, as many factors as possible affecting microglial functions in in vitro culture systems are presented in this review. The factors are separated into "activators" and "inhibitors," although it is difficult to classify many of them. An overview on these factors may help in the development of a new strategy for the treatment of various neurodegenerative diseases.
Neuroscience Research Supplements
Tight-seal whole-cell clamp experiments were performed on thin slices (150-250 p) obtained from t... more Tight-seal whole-cell clamp experiments were performed on thin slices (150-250 p) obtained from the caudate nucleus of rats (9-14 days old). Most of the small cells (lo-15 w in diameter) stained with Lucifer Yellow exhibited long dendrites stubbed with fine spines, which is characteristic of medium-sized spiny neurons. Focal stimulation of the caudate nucleus with a glass pipette purely evoked excitatory synaptic currents in the presence of picrotoxin. The EPSCs were composed of NMDA and non-NMDA components; APV abolished the former component and CNQX the latter. Spontaneous EPSCs also comprised the two components, suggesting co-localization of the two receptor subtypes in the postsynaptic membranes. One feature of the ghttamatergic synaptic transmission was that, in the paired-pulsed experiments, the second EPSC was inhibited to 30% of the first EPSCs. This marked paired-pulse inhibition occurred both in the NMDA and non-NMDA components, showed no delay in onset and persisted up to 500 ms in the NMDA components, but only to 30 ms in the non-NMDA component. Quantal analysis suggested the involvement of a presynaptic mechanism in the inhibition of the non-NMDA component.
Neurochemistry International
Microglial activation has been suggested to play important roles in various neurodegenerative dis... more Microglial activation has been suggested to play important roles in various neurodegenerative diseases by phagocytosis and producing various factors such as nitric oxide (NO), proinflammatory cytokines. Excessive production of NO, as a consequence of increased inducible nitric oxide synthase (iNOS) in microglia, contributes to the neurodegeneration. During a search for compounds that regulate endoplasmic reticulum (ER) stress, a dibenzoylmethane derivative, 2,2'-dimethoxydibenzoylmethane (DBM 14-26) was identified as a novel neuroprotective agent (Takano et al., Am. J. Physiol. Cell Physiol. 293, C1884-1894, 2007). We previously reported in cultured astrocytes that DBM 14-26 protected hydrogen peroxide-induced cell death and inhibited lipopolysaccharide (LPS)-induced NO production (Takano et al., J. Neurosci. Res. 89, 955-965, 2011). In the present study, we assessed the effects of DBM 14-26 on microglia using the mouse cell line BV-2 and found that DBM 14-26 inhibited LPS-induced iNOS expression and NO production also in microglia. DBM 14-26 also suppressed LPS-induced IL-1β expression. Conditioned medium of BV-2 cells stimulated by LPS significantly decreased cell viability of neuron (human neuroblastoma SH-SY5Y cells) compared with the absence of LPS. Conditioned medium of BV-2 cells stimulated by LPS in the presence of DBM 14-26 did not significantly decreased cell viability of neuron. These results indicate that microglial activation by LPS causes neuronal cell death and DBM 14-26 protect neuron through the inhibition of microglial activation. Functional regulation of microglia by DBM 14-26 could be a therapeutic candidate for the treatment of neurodegenerative diseases.
Neurochem Res, 1994
From rat hippocampal homogenate, we recently isolated a novel subcellular fraction richly contain... more From rat hippocampal homogenate, we recently isolated a novel subcellular fraction richly containing glial plasmalemmal vesicles (GPV), which takes up glutamate remarkably as a synaptosomal fraction [Y. Nakamura et al. (1993) Glia, 9, 48-56]. In the present study, we prepared GPV from different regions of rat CNS, namely olfactory bulb (Ob), cerebral cortex (Cx), caudatoputamen (Cp), hippocampus (Hp), cerebellum (Ce) and spinal cord (Sc), and analyzed their activities of Na+-dependent uptake of following neurotransmitters and a related compound; glutamate, ",/-aminobutyrate (GABA), glycine, dopamine and choline. The uptake activities of these amino acids were not significantly different between GPV and synaptosomes in each region. Regionally, however, the activities were varied considerably. The activities of glutamate uptake revealed in the following rank order: Cx, Hp, Cp > Ce, Ob > Sc. GABA uptake activities were: Ce > Ob, Cx, Hp > Cp, while glycine uptake activities were: Sc, Ce > Ob, Cp, Cx, Hp. On the other hand, the uptake activities of dopamine and choline were quite different between GPV and synaptosomes. Synaptosomal fraction from Cp took up dopamine in a high activity; however, GPV from the same tissue hardly showed the uptake activity. Choline was taken up by synaptosomes prepared from Hp but not by GPV.
J Neurochem, 2002
The time course of the decline in energy levels during an in vitro ischemia-like condition was co... more The time course of the decline in energy levels during an in vitro ischemia-like condition was compared with changes in intracellular Ca2+ concentration ([Ca2+]i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod-2. During the on-line monitoring of [Ca2+]i, the slices were perfused with an in vitro ischemia-like medium (33 degrees C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region-specific and acute [Ca2+]i elevations were observed in CA1 approximately 4 min after onset of the in vitro ischemia-like condition and also in the inner portion of the DG with a delay of 10-40 s. The change in ATP levels was related to the increase in [Ca2+]i. ATP levels in all subregions gradually decreased before the acute [Ca2+]i elevation. Concomitant with the acute [Ca2+]i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high-energy-charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca2+]i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region-specific increase in [Ca2+]i, which activates a Ca(2+)-ATP-driven pump and produces a subsequent fall in neuronal ATP content.
Journal of Biochemistry, Feb 1, 1978
Neurochem Res, 1995
In order to elucidate the mechanism of release of excitatory amino acid (EAA) induced by hypoxia-... more In order to elucidate the mechanism of release of excitatory amino acid (EAA) induced by hypoxia-hypoglycemia (in vitro ischemia) from cultured hippocampal astrocytes, we compared the EAA release by in vitro ischemia with those by other treatments. The EAA release induced by in vitro ischemia treatment was rapid and reversible. The amount of released aspartate was comparable to that of glutamate, although the endogenous content of aspartate was one sixth that of glutamate. High-K (100 mM) treatment and the addition of 5 mM NaCN induced a rapid EAA release and the glutamate release was much greater than aspartate. Addition of 5 mM iodoacetate, a glycolysis inhibitor, induced a slow EAA release, and the amount of released aspartate was much higher than that of glutamate. On the other hand, the in vitro ischemia treatment and the addition of 5 mM NaCN induced only 20% reduction in ATP content for initial 5 min, whereas the addition of 5 mM iodoacetate induced a marked reduction. Our data suggest that ischemia-induced EAA release from astrocytes is a complex process in which local energy failure, inhibition of glycolysis, and depolarization of the cell membrane are involved.
Endocrinology, Dec 1, 1986
Microsomal fractions prepared from porcine thyroid glands by differential centrifugations and suc... more Microsomal fractions prepared from porcine thyroid glands by differential centrifugations and sucrose density gradient centrifugation showed an ATP-dependent Ca2+ uptake. Electron microscopy and the study of marker enzyme activities suggested that the fractions consisted mainly of endoplasmic reticulum. The amount of transported Ca2+ increased four times in the presence of 20 mM oxalate owing to the precipitation of calcium oxalate, which was detected inside the microsomal vesicles by electron microscopy. Ca2+ was released rapidly when the calcium ionophore, A-23187, was added. The Ca2+ concentration for the half-maximal activation of Ca2+ transport was about 1 microM. These results indicate that Ca2+ is translocated into the lumen of microsomes against a concentration gradient in a manner of the active transport. The microsomes showed Ca2+-dependent ATPase activity and were phosphorylated by the reaction with [gamma-32P]ATP in a similar Ca2+ dependence to that of transport rate. A 105-kDa phosphoprotein was identified by dodecyl sulfate polyacrylamide gel electrophoresis and was found to be sensitive to hydroxylamine. These properties of the phosphoprotein were the same as those of Ca2+ pump ATPase in the endoplasmic reticulum of other cells. These results suggest that the cytosolic Ca2+ is maintained at low levels by the microsomal uptake of Ca2+ by the action of the ATP-dependent Ca2+ pump or active transport system.
Brain Res, 2003
Microglial proliferation and activation have been reported to occur after several central nervous... more Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10-300 microM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P(2)y receptor agonists (ADPbetaS and 2-methylthio-ATP) on LPS-induced TNF-alpha release. The rank order of inhibitory potency of endogenous purines on TNF-alpha release was: ATP>ADP>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 microM 2-methylthio-ATP showed a potency similar to 100 microM ATP. The addition of ADPbetaS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P(2)y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.
J Neurochem, 2008
Microglial activation has recently been recognized as a cause of damage in various neurodegenerat... more Microglial activation has recently been recognized as a cause of damage in various neurodegenerative diseases. A possible mechanism underlying this damage is the activation of microglia by serum factors leaked through a disruption of the blood-brain barrier, which in turn trigger microglial cell proliferation and the release of various substances toxic to neurons, such as superoxide (O(2)(-)). We recently reported that serum albumin enhanced O(2)(-) production in cultured rat microglia stimulated by phorbol ester. In the present report, we identify the active site of this enhancement within the albumin molecule. We purified an active subfragment from trypsin-treated bovine serum albumin that was composed of 12-mer and 33-mer peptides connected by a disulfide bond. The chemically synthesized 12-mer peptide showed activity within a concentration range ( approximately 10(-7) M:) equivalent to that of albumin. The activities of a series of synthesized peptides conclusively indicated that the minimum active sequence was Leu-His-Thr-Leu. The present study may shed light on the mechanism of neuronal cell damage in various neurodegenerative diseases.
Structure and Function of Sarcoplasmic Reticulum, 1985
NeuroImmune Biology, 2008
Neuroscience Research Supplements, 1994
Biochimica et biophysica acta, Jan 5, 2015
The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-pho... more The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusio...
Neuroimmunomodulation, Jan 3, 2014
Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be invol... more Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). Methods: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RT-PCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-µm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. Results: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulat...
The Journal of veterinary medical science / the Japanese Society of Veterinary Science, 2014
Amphotericin B (AmB) is a polyene antifungal drug and is reported to be one of a few reagents hav... more Amphotericin B (AmB) is a polyene antifungal drug and is reported to be one of a few reagents having therapeutic effects on prion diseases, that is, a delay in the appearance of clinical signs and prolongation of the survival time in an animal model. In prion diseases, glial cells have been suggested to play important roles; however, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotrophic factors in microglia and astrocytes (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290-1296; Motoyoshi-Yamashiro et al., 2013, ibid. 63, 93-100). These results suggested that neurotrophic factors derived from glial cells might be involved in the therapeutic mechanism of AmB. In the present study, we examined immunohistochemically the effects of AmB on the expression of neurotrophic factors in the rat brain. We found that direct injection of AmB into the striatum significantly enhanced the expression of glial cell...
Neurochemistry international, 2006
Uptake of K+ is an important role of astrocytes to maintain physiological lower extracellular K+ ... more Uptake of K+ is an important role of astrocytes to maintain physiological lower extracellular K+ concentration in the CNS. In this study, the effect of high K+ concentration was examined on the cellular function of astrocytes from embryonic rat brain in primary culture. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) was measured as an index of cellular function of astrocytes. Increasing KCl concentration to about 40 mM did not directly evoke NO production, but doubled the level of LPS (1 ng/ml)-induced NO production. K-gluconate showed a similar enhancing effect although the degree of enhancement was about half of that of KCl. Neither NaCl nor Na-gluconate showed any effect. The K(+)-channel blocker, 4-aminopyridine, but not tetraethylammonium or apamin, inhibited the enhancing effect of KCl. The LPS-induced iNOS protein expression determined by immunoblotting analysis was enhanced by high K+ treatment. The level of iNOS mRNA determined by real-time RT-PCR techniqu...
Current neurovascular research, 2005
The polyamines, putrescine, spermidine and spermine are present in most living cells, with the es... more The polyamines, putrescine, spermidine and spermine are present in most living cells, with the essentiality for normal cell function, cellular growth and differentiation. In the mammalian brain, polyamines are also present at relatively high concentrations with different regional distribution profiles. Cerebral ischemia is a leading cause of disability and mortality in humans, and believed to yield a cascade of cytotoxic molecules responsible for the death of viable cells in the brain. Polyamines have been implicated in the pathogenesis of ischemic brain damage. For example, polyamine biosynthesis is increased after the onset of cerebral ischemia through an induction of ornithine decarboxylase, a key enzyme in the polyamine biosynthetic pathway. The administration of a drug that inhibits ornithine decarboxylase activity prevents the development of ischemic brain damage, suggesting a critical role of the accumulation of polyamines in the ischemic brain in the pathogenesis of stroke. ...