Kentaro Nishida

Kyoto Pharmaceutical University, Kioto, Kyoto, Japan

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Publications (24)71.13 Total impact

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    ABSTRACT: Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5 mM ATP for 2 h, followed by 3 h-incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.
    No preview · Article · Jan 2016 · Neuroscience
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    ABSTRACT: To elucidate the regulatory mechanism for extracellular zinc in the CNS, we examined the zinc uptake characteristics in mouse astrocytes and microglia. Zinc was taken up into the two cell types time-dependently, and the cell-to-medium concentration (C/M) ratios in the initial uptake phase in astrocytes was significantly smaller than that in microglia, while in the steady state phase, there was no difference in their C/M ratios. In both astrocytes and microglia, the zinc uptake was mediated, at least in part, by high- and low-affinity systems. There were no differences for both in the Km values for zinc uptake between astrocytes and microglia, and those for the low-affinity system in both cell types were the same as that for mouse ZIP1 reported previously. On the other hand, the Vmax values for both systems were greater in microglia than in astrocytes. Among ZIP isoforms, expression of ZIP1 was high in astrocytes and microglia. Nickel, a competitive inhibitor of ZIP1, and ZIP1 knockdown decreased zinc uptake by both types of cells. Overall, it is demonstrated that astrocytes and microglia had the similar uptake system for zinc including ZIP1, and the differences found in their uptake profiles imply that they play different roles in regulation of extracellular zinc to maintain brain homeostasis.
    No preview · Article · Apr 2015 · Metallomics
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    ABSTRACT: We previously demonstrated that the P2X7 receptor (P2X7R), a purinergic receptor, expressed by mouse cultured cortical astrocytes is constitutively activated without any exogenous stimulus, differing from the case of neurons. It is well known that astrocytic morphology differs between in vitro and in vivo situations, implying different functionalities. Brain acute slices are widely accepted as an in vitro experimental system that reflects in vivo cell conditions better than in vitro cell culture ones. We examined whether astrocytic P2X7Rs exhibited constitutive activation in mouse cortical slices. In acute cortical slices, P2X7R-immunoreactivity was detected in both glial fibrillary acidic protein-immunopositive astrocytes and microtubule-associated protein 2-immunopositive neurons. Astrocytic, but not neuronal, spontaneous uptake of propidium iodide, an indicator of P2X7R channel/pore activity, was inhibited by representative antagonists of P2X7R, but they had no effect on the uptake by astrocytes in membrane-permeabilized fixed slices. These findings indicate that astrocytes, but not neurons, in acute cortical slices exhibit constitutive activation of P2X7Rs under non-stimulated resting conditions as in the case of cell culture systems.
    No preview · Article · Dec 2014 · Biological & Pharmaceutical Bulletin
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    ABSTRACT: Aim In this study, we evaluated the effect of divalent metal cations on zinc uptake via mouse Zrt/Irt-like protein 1 (mZIP1), a ubiquitously expressed zinc transporter, which plays a role in cellular zinc homeostasis, using HEK293T cells overexpressing it. Main methods mZIP1 cDNA, which was cloned from mouse microglia, was transfected into HEK293T cells by a lipofection method, and its functional expression was confirmed by Western blotting and immunocytochemical analyses, and 65Zn (65ZnCl2) uptake. Key findings 65Zn uptake by mZIP1 cDNA-transfected cells time-dependently increased compared with that by mock cells, indicating functional expression of mZIP1. mZIP1-mediated 65Zn uptake showed clear saturable kinetics consisting of a single component with a Michaelis constant of 5.88 μM. FeCl2 and NiCl2 competitively inhibited the 65Zn uptake, the inhibition constants (Ki) being estimated to be 0.92 and 28.6 μM, respectively. In addition, CoCl2 and CdCl2 showed non-competitive inhibition of mZIP1-mediated 65Zn uptake, the Ki values being 219 and 32.5 μM, respectively. On the other hand, CuCl2 also significantly decreased the uptake, but the inhibition mode could not evaluate because of its low solubility, while MnCl2 and MgCl2 had no effect on 65Zn uptake via mZIP1. Significance Iron, nickel, cobalt and cadmium, in addition to zinc, act as inhibitors of mZIP1, the affinity order being iron > zinc > nickel = cadmium > cobalt, and copper might also act as an inhibitor, while manganese and magnesium are not recognized by mZIP1. These findings provide valuable information as to the contribution of mZIP1 to total cellular zinc transport.
    No preview · Article · Sep 2014 · Life Sciences
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    ABSTRACT: ATP plays an important role in the mechanism of signal transduction between sensory neurons and satellite cells in dorsal root ganglia (DRGs). In primary cultured DRG neurons, ATP is known to be stored in lysosomes via a vesicular nucleotide transporter (VNUT), and to be released into the intercellular space through exocytosis. DRGs consist of large-, medium- and small-sized neurons, which play different roles in sensory transmission, but there is no information on the expression profiles of VNUT in DRG subpopulations. Here, we obtained detailed expression profiles of VNUT in isolated rat DRG tissues. On immunohistochemical analysis, VNUT was found in DRG neurons, and was predominantly expressed by the small- and medium-sized DRG ones, as judged upon visual inspection, and this was compatible with the finding that the number of VNUT-positive DRG neurons in IB4-positive cells was greater than that in NF200-positive ones. These results suggest that VNUT play a role in ATP accumulation in DRG neurons, especially in small- and medium-sized ones, and might be involved in ATP-mediated nociceptive signaling in DRGs.
    No preview · Article · Jul 2014 · Neuroscience Letters
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    ABSTRACT: Of purinergic receptors, P2X7 receptor (P2X7R, defined as a full-length receptor) has unique characteristics, and its activation leads to ion channel activity and pore formation, causing cell death. Previously, we demonstrated that P2X7R expressed by nonstimulated astrocyte cultures obtained from SJL-strain mice exhibits constitutive activation, implying its role in maintenance of cellular homeostasis. To obtain novel insights into its physiological roles, we examined whether constitutive activation of P2X7R is regulated by expression of its splice variants in such resting astrocytes, and whether their distinct expression profiles in different mouse strains affect activation levels of astrocytic P2X7Rs. In SJL- and ddY-mouse astrocytes, spontaneous YO-PRO-1 uptake, an indicator of pore activity of P2X7R, was detected, but the uptake by the formers was significantly greater than that by the latter. Between the two mouse strains, there was a difference in their sensitivity of YO-PRO-1 uptake to antagonists, but not in the expression levels and sequences of P2X7R and pannexin-1. Regarding expression of splice variants of P2X7R, expression of P2X7R variant-3 (P2X7R-v3) and -4 (P2X7R-v4), but not variant-2 and -k, was lower in SJL-mouse astrocytes than in ddY-mouse ones. On transfection of P2X7R-v3 and -v4 into SJL-mouse astrocytes, the pore activity was attenuated as in the case of the HEK293T cell-expression system. These findings demonstrate that basal activity of P2X7R expressed by resting astrocytes is negatively regulated by P2X7R-v3 and -v4, and that their distinct expression profiles result in the different activation levels of astrocytic P2X7Rs in different mouse strains. GLIA 2013;
    No preview · Article · Mar 2014 · Glia
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    ABSTRACT: We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.
    No preview · Article · Dec 2013 · Histochemie
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    ABSTRACT: Astrocytes contribute to maintenance of brain homeostasis via release of gliotransmitters such as ATP and glutamate. Here we examined whether zinc was released from astrocytes under stress-loaded conditions, and was involved in regulation of microglial activity as a gliotransmitter. Hypoosmotic stress was loaded to astrocytes using balanced salt solution prepared to 214-314 mOsmol/L, and then intra- and extra-cellular zinc levels were assessed using Newport Green DCF diacetate (NG) and ICP-MS, respectively. Microglial activation by the astrocytic supernatant was assessed by their morphological changes and poly(ADP-ribose) (PAR) polymer accumulation. Exposure of astrocytes to hypoosmotic buffer, increased the extracellular ATP level in osmolarity-dependent manners, indicating load of hypoosmotic stress. In hypoosmotic stress-loaded astrocytes, there were apparent increases in the intra- and extra-cellular zinc levels. Incubation of microglia in the astrocytic conditioned medium transformed them into the activated "amoeboid" form and induced PAR formation. Administration of an extracellular zinc chelator, CaEDTA, to the astrocytic conditioned medium almost completely prevented the microglial activation. Treatment of astrocytes with an intracellular zinc chelator, TPEN, suppressed the hypoosmotic stress-increased intracellular, but not the extracellular, zinc level, and the increase in the intracellular zinc level was blocked partially by a nitric oxide synthase inhibitor, but not by CaEDTA, indicating that the mechanisms underlying the increases in the intra- and extra-cellular zinc levels might be different. These findings suggest that under hypoosmotic stress-loaded conditions, zinc is released from astrocytes and then plays a primary role in microglial activation as a gliotransmitter.
    No preview · Article · Nov 2013 · Life sciences
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    ABSTRACT: We previously demonstrated that P2X7 receptors (P2X7Rs) expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but its roles in non-stimulated resting astrocytes remained unknown. It has been reported that astrocytes exhibit engulfing activity, and that the basal activity of P2X7Rs regulates the phagocytic activity of macrophages. In this study, therefore, we investigated whether P2X7Rs regulate the engulfing activity of mouse astrocytes. Uptake of non-opsonized beads by resting astrocytes derived from ddY-mouse cortex time-dependently increased, and the uptaken beads were detected in the intracellular space. The bead uptake was inhibited by cytochalasin D (CytD), an F-actin polymerization inhibitor, and agonists and antagonists of P2X7Rs apparently decreased the uptake. Spontaneous YO-PRO-1 uptake by ddY-mouse astrocytes was reduced by the agonists and antagonists of P2X7Rs, but not by CytD. Down-regulation of P2X7Rs using siRNA decreased the bead uptake by ddY-mouse astrocytes. In addition, compared to in the case of ddY-mouse astrocytes, SJL-mouse astrocytes exhibited higher YO-PRO-1 uptake activity, and their bead uptake was significantly greater. These findings suggest that resting astrocytes exhibit engulfing activity and that the activity is regulated, at least in part, by their P2X7Rs.
    No preview · Article · Aug 2013 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Aim: Cell death induced by excessive activation of poly(ADP-ribose) polymerase (PARP) is inhibited by administration of NAD(+) extracellularly, but its preventive mechanism remains unclear. Here we investigated the involvement of NAD(+) and/or its metabolites, adenosine and nicotinamide, in the rescue of PARP-mediated astrocyte death by NAD(+) and explored the pathway through which intact NAD(+) could enter cells. Main methods: PARP activation was induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent. The cellular NAD(+) content was determined by an enzymatic recycling assay, and cell viability was determined by measuring intracellular LDH activity. Key findings: NAD(+), but not adenosine and nicotinamide, could restore the cellular NAD(+) levels decreased by PARP activation. Pharmacological inhibition of the uptake of adenosine and nicotinamide had no effect on the prevention of PARP-triggered cell death by NAD(+), suggesting that unmetabolized NAD(+) remaining in the extracellular milieu might prevent PARP-mediated NAD(+) consumption and cell death. The increase in the cellular NAD(+) level caused by NAD(+) administration to PARP-activated cells was significantly inhibited by a connexin hemichannel blocker, carbenoxolone, but not by P2X7 receptor inhibition with selective antagonists and siRNA, or pannexin-selective blockers. Finally, pharmacological blockade of connexin hemichannels with 18β-glycyrrhetinic acid, octanol and carbenoxolone inhibited the NAD(+)-mediated cell rescue of PARP-triggered cell death. Significance: These findings suggested that intact NAD(+) could get into astrocytes through connexin hemichannels, and that this process should play a key role in NAD(+)-mediated prevention of PARP-triggered astrocyte death.
    No preview · Article · Feb 2013 · Life sciences
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    ABSTRACT: In gustatory function, communication between four types taste buds cells plays crucial roles. ATP is one of the intercellular signaling molecules in taste buds, and the extracellular ATP fate is regulated by its cellular clearance, but there is little information on it. Therefore, we examined the expression profiles of nucleoside transporters (NTs) as a clearance system for ATP metabolite adenosine in rat circumvallate papillae (CP) by RT-PCR, real-time PCR and immunohistochemistry. Among NTs, mRNA for Ent1 was expressed by the CP, and significantly increased in the CP as compared with non-CP. ENT1 immunoreactivity was detected in PLC-β2-positive type II (71.0±8.5%), chromogranin-A-positive type III (64.9±7.4%), and SNAP25-positive type III (77.0±10.4%) taste cells, but not in NTPDase2-positive type I ones. These results indicate that ENT1-expressing type II and III taste cells might comprise an adenosine clearance system in taste buds of the CP. ENT1 expression in taste cells is important for elucidation of complicated taste signaling.
    No preview · Article · Nov 2012 · Neuroscience Letters
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    ABSTRACT: J. Neurochem. (2012) 122, 1118–1128. P2X7 receptor (P2X7R) is known to be a ‘death receptor’ in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca2+ entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca2+ entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.
    Full-text · Article · Jul 2012 · Journal of Neurochemistry
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    ABSTRACT: Previously, we demonstrated that extracellular zinc plays a key role in transient global ischemia-induced microglial activation through sequential activation of NADPH oxidase and poly(ADP-ribose) polymerase (PARP)-1. However, it remains unclear how zinc causes the sequential activation of microglia. Here, we examined whether transporter-mediated zinc uptake is necessary for microglial activation. Administration of zinc to microglia activated them through reactive oxygen species (ROS) generation and poly(ADP-ribose) (PAR) formation, which were suppressed by intracellular zinc chelation with 25 μM TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) or 2 μM BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester). The 65Zn uptake by microglia was temperature- and dose-dependent, and it was blocked by metal cations, but not by L-type calcium channel blockers nifedipine and nimodipine. Expression of Zrt-Irt-like protein (ZIP)1, a plasma membrane-type zinc transporter, was detected in microglia, and nickel, a relatively sensitive substrate/inhibitor of ZIP1, showed cis- and trans-inhibitory effects on the 65Zn uptake. Exposure of microglia to zinc increased the extracellular ATP concentration, which was suppressed by intracellular zinc chelation and inhibition of hemichannels. mRNA expression of several types of P2 receptors was detected in microglia, and periodate-oxidized ATP, a selective P2×7 receptor antagonist, attenuated the zinc-induced microglial activation via NADPH oxidase and PARP-1. Exogenous ATP and 2′(3′)-O-(4-benzoyl-benzoyl) ATP also caused microglial activation through ROS generation and PAR formation. These findings demonstrate that ZIP1-mediated uptake of zinc induces ATP release and autocrine/paracrine activation of P2X(7) receptors, and then activates microglia, suggesting that zinc transporter-mediated uptake of zinc is a trigger for microglial activation via the NADPH oxidase and PARP-1 pathway.
    No preview · Article · Dec 2011 · Glia
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    ABSTRACT: In the oxidative stress-loaded brain, extracellular adenosine levels are elevated and thereby neuronal damage is attenuated, but mechanisms underlying alteration of the extracellular kinetics of adenosine remain unclear. Here we investigated whether oxidative stress might alter functional expression of nucleoside transporters (NTs), a predominant regulatory system for nucleoside kinetics, in cultured rat astrocytes. Treatment of astrocytes with 0.5mM SIN-1 for 3h caused apparent cellular accumulation of nitrotyrosine, but had no effect on their viability, indicating load of oxidative stress to astrocytes without any change in their viability. Under the condition, [(3)H]adenosine uptake was significantly less than that by control cells. This decreased uptake was due to decrease in adenosine uptake mediated by an equilibrative NT (ENT) 1 which was inhibited by low concentrations (≤0.1 μM) of nitrobenzylthioinosine (NBMPR), but not by sodium-dependent or high concentrations (≥1 μM) of NBMPR-inhibitable nucleoside transporters. The expression level of ENT1 was not altered, while the Michaelis constant, but not the maximum rate, of adenosine uptake was increased. These findings suggest that under oxidative stress-loaded conditions, decreased adenosine clearance via astrocytic ENT1 might involve, at least in part, in an elevated extracellular adenosine level in the brain.
    No preview · Article · Jul 2011 · Neuroscience Letters
  • Hiroto Okuda · Yuka Tokotani · Kentaro Nishida · Kazuki Nagasawa

    No preview · Article · Dec 2010 · Neuroscience Research

  • No preview · Article · Dec 2010 · Neuroscience Research
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    ABSTRACT: Nucleotides and nucleosides play important roles by maintaining brain homeostasis, and their extracellular concentrations are mainly regulated by ectonucleotidases and nucleoside transporters expressed by astrocytes. Extracellularly applied NAD(+) prevents astrocyte death caused by excessive activation of poly(ADP-ribose) polymerase-1, of which the molecular mechanism has not been fully elucidated. Recently, exogenous NAD(+) was reported to enter astrocytes via the P2X7 receptor (P2X7R)-associated channel/pore. In this study, we examined whether the intact form of NAD(+) is incorporated into astrocytes. A large portion of extracellularly added NAD(+) was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring-labeled [(14)C]NAD(+), but not nicotinamide moiety-labeled [(3)H]NAD(+), showed time- and temperature-dependency, and was significantly enhanced on addition of apyrase, and was reduced by 8-Br-cADPR and ARL67156, inhibitors of CD38 and ectoapyrase, respectively, and P2X7R knockdown, suggesting that the detected uptake of [(14)C]NAD(+) resulted from [(14)C]adenosine acting as a metabolite of [(14)C]NAD(+). Pharmacological and genetic inhibition of P2X7R with brilliant blue G, KN-62, oxATP, and siRNA transfection resulted in a decrease of [(3)H]adenosine uptake, and the uptake was also reduced by low concentration of carbenoxolone and pannexin1 selective peptide blocker (10)panx. Taken together, these results indicate that exogenous NAD(+) is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R-associated channel/pore.
    No preview · Article · Nov 2010 · Glia
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    ABSTRACT: Concentrative nucleoside transporter 1 (CNT1, SLC28A1) is a key molecule for determining the pharmacokinetic/pharmacodynamic profile of a candidate compound derived from a pyrimidine nucleoside, but there is no available information on the differences in the functional profile of this ortholog between man and mouse. Here, using a clone of mouse CNT1 (mCNT1), we investigated its transport characteristics and substrate specificity for synthetic nucleoside analogues, and compared them with those of human CNT1 (hCNT1). In mCNT1-transfected Cos-7 cells, pyrimidine, but not purine, nucleosides showed sodium- and concentration-dependent uptake, and uridine uptake was competitively inhibited by uridine analogues, the rank order of the inhibitory effects being 5-bromouridine>3'-deoxyuridine>2'-deoxyuridine. cis- and trans-Inhibition studies involving synthetic nucleoside drugs revealed that gemcitabine and zidovudine greatly inhibited [(3)H]uridine uptake mediated by mCNT1 in the both cases, while cytarabine and zalcitabine showed small cis-inhibitory effect, and no trans-inhibitory effect on the uptake. These results demonstrate that the transport characteristics of mCNT1 are almost the same as those of hCNT1, suggesting that mice may be a good animal model in evaluation of pyrimidine nucleoside analogues as to their applicability in human therapy.
    No preview · Article · Mar 2010 · International Journal of Pharmaceutics
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    ABSTRACT: Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme, and its excessive activation, following ischemia, trauma, etc., depletes cellular nicotinamide adenine dinucleotide (NAD(+)) as a substrate and eventually leads to brain cell death. Nicotinamide, an NAD(+) precursor and a PARP-1 inhibitor, is known to prevent PARP-1-triggered cell death, but there is no available information on the mechanisms involved in its transport. Here we clarified the transport characteristics of nicotinamide in primary cultured mouse astrocytes. Uptake characteristics of [(14)C]nicotinamide were assessed by a conventional method with primary cultured mouse astrocytes. Cell viability and PARP-1 activity were determined with intracellular LDH activity and immunocytochemical detection of PAR accumulation, respectively. PARP-1 activation was induced by treatment of astrocytes with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent. MNNG-triggered astrocyte death and PAR accumulation were completely inhibited by treatment with nicotinamide as with DPQ (3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone), a second generation PARP inhibitor. The uptake of [(14)C]nicotinamide was time-, temperature-, concentration- and pH-dependent, and was inhibited and stimulated by co- and pre-treatment with N-methylnicotinamide, a representative substrate of an organic cation transport system, respectively. Co-treatment of astrocytes with nicotinamide and N-methylnicotinamide resulted in a decrease in PAR accumulation and absolute prevention of cell death. These findings suggest that nicotinamide has a protective effect against PARP-1-induced astrocyte death and that its transporter-mediated uptake, which is extracellular pH-sensitive and common to N-methylnicotinamide, is critical for prevention of PARP-1-triggered cell death.
    No preview · Article · Feb 2010 · Life sciences
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    ABSTRACT: Recently, we demonstrated that cultured mouse astrocytes exhibited basal channel opening of P2X7 receptor (P2X7R) in the absence of any exogenous ligand, but the regulatory mechanism involved was not elucidated. Since our preliminary experiments suggested possible involvement of peroxisome proliferator-activated receptor (PPAR) gamma in the regulation, we examined whether PPAR gamma regulated P2X7R basal channel opening in mouse astrocytes. P2X7R channel opening was assessed as to the uptake of a marker dye, YO-PRO-1 (YP), in the presence or absence of agonists and antagonists for PPAR gamma under a fluorescence microscope. Expression of PPAR gamma was evaluated by Western blotting and immunocytochemistry. NSAIDs such as flufenamic acid (FFA) and indomethacin, which are a cyclooxygenase inhibitor and a PPAR gamma agonist, showed enhancing and inhibiting effects on YP uptake at low and high concentrations, respectively, and the enhanced uptake was abolished by periodate-oxidized ATP (oxATP), a selective P2X7R antagonist. The PPAR gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) and ciglitazone decreased the basal and FFA-enhanced YP uptake, while the antagonist GW9662 increased YP uptake, this effect being blocked by the agonists and also by oxATP. PPAR gamma was distributed in the nucleus and cytosolic/membrane fraction of cultured mouse astrocytes. These findings indicate that basal channel opening of P2X7R in mouse astrocytes is at least in part regulated by PPAR gamma.
    No preview · Article · May 2009 · Life sciences

Publication Stats

129 Citations
71.13 Total Impact Points


  • 2004-2016
    • Kyoto Pharmaceutical University
      • Laboratory of Environmental Biochemistry
      Kioto, Kyoto, Japan
  • 2010
    • Kyoto University
      Kioto, Kyōto, Japan