Kentaro Nishida

Microglial zinc uptake via zinc transporters induces ATP release and the activation of microglia

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.

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death.

J. Neurochem. (2012) 122, 1118–1128.

P2X7 receptors regulate engulfing activity of non-stimulated resting astrocytes.

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.

Contribution of P2X7 receptors to adenosine uptake by cultured mouse astrocytes

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 [14C]NAD+, but not nicotinamide moiety‐labeled [3H]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 [14C]NAD+ resulted from [14C]adenosine acting as a metabolite of [14C]NAD+. Pharmacological and genetic inhibition of P2X7R with brilliant blue G, KN‐62, oxATP, and siRNA transfection resulted in a decrease of [3H]adenosine uptake, and the uptake was also reduced by low concentration of carbenoxolone and pannexin1 selective peptide blocker 10panx. 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.

Regulation of activity of P2X7 receptor by its splice variants in cultured mouse astrocytes

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 2014;62:440–451

Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons

ATP plays an important role in the 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.

Possible involvement of PPARγ in the regulation of basal channel opening of P2X7 receptor in cultured mouse astrocytes

AIMS 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. MAIN METHODS 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. KEY FINDINGS 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. SIGNIFICANCE These findings indicate that basal channel opening of P2X7R in mouse astrocytes is at least in part regulated by PPAR gamma.

Oxidative stress upregulates zinc uptake activity via Zrt/Irt-like protein 1 (ZIP1) in cultured mouse astrocytes.

AIMS Zinc released from glutamatergic boutons and astrocytes acts as neuro- and glio-transmitters, and thus its extracellular level has to be strictly regulated. We previously revealed that uptake of zinc by astrocytes plays a critical role in its clearance, and zinc transporter Zrt/Irt-like protein 1 (ZIP1) is the molecule responsible for the uptake. However, it is unknown whether or not the functionality of the zinc clearance system is altered under oxidative stress-loaded conditions. Here, we characterized zinc uptake by oxidative stress-loaded astrocytes. MAIN METHODS Cultured mouse astrocytes were treated with hydrogen peroxide (H2O2) to load oxidative stress. Functional expression of ZIP1 in astrocytes was evaluated by means of (65)Zn uptake, Western blotting and immunocytochemical analysis. KEY FINDINGS Treatment of astrocytes with 0.4mM H2O2 for 24h increased the expression levels of glial fibrillary acidic protein and 4-hydroxynonenal without significant decreases in their viability, indicating that induction of oxidative stress in astrocytes. Under oxidative stress-loaded conditions, astrocytes exhibited increased (65)Zn uptake activity, and the maximum uptake velocity for the uptake was significantly increased compared to that in the control group, while there was no change in the Michaelis constants, which were almost identical to that of mouse ZIP1. In the H2O2-treated astrocytes, the expression levels of ZIP1 were significantly increased in the cellular and plasma membrane fractions. SIGNIFICANCE It appears that under oxidative stress-loaded conditions, astrocytes exhibit increased zinc clearance activity and this is due, at least in part, to increased ZIP1 expression.

Peroxynitrite treatment reduces adenosine uptake via the equilibrative nucleoside transporter in rat astrocytes.

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.

Expression of equilibrative nucleoside transporter 1 in rat circumvallate papillae.

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 was greater 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.