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

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 Ca(2+) 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 Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.

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.

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 [(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.

Protective effect of nicotinamide against poly(ADP-ribose) polymerase-1-mediated astrocyte death depends on its transporter-mediated uptake.

AIM: 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. MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.

Transport characteristics of mouse concentrative nucleoside transporter 1.

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.

Possible involvement of PPAR gamma 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.

Altered levels of oxidation and phospholipase C isozyme expression in the brains of theanine-administered rats.

Previously, we revealed that theanine, a green tea component, induced phospholipase C (PLC)-beta1 and -gamma1, stress-responsible molecules, in primary cultured rat cerebral cortical neurons, suggesting its protective effect on oxidative stress in neurons. In this study, we investigated whether the same favorable effect occurs in vivo. On the oral administration of theanine (10 mmol (1.74 g)/kg, once a day) to rats via gastric intubation for 2 weeks, there was no change in the weight of the body or the cerebral cortex (Cx), cerebellum (Cb), or hippocampus (Hip) in the brain. On assessment of oxidation levels in the brain with thiobarbiturate reactive substances as a marker, the levels were found to be 20% lower in the Cx of theanine-treated rats than in that of control ones. The protein expression levels of PLC-beta1 and -gamma1 were significantly increased in the Cx on theanine administration and the same tendency was observed in the Cb, but not the Hip. In addition, the protein expression level of PLC-delta1, which plays an opposite role to the other two isozymes, was not affected in any brain regions on theanine administration. Overall, it was demonstrated that theanine is a safe compound and its repeated oral administration reduces oxidation levels in the brain, especially the Cx, by increasing PLC-beta1 and -gamma1 protein expression, suggesting its favorable effect on the brain in vivo.

Decreased expression of phospholipase C-beta 1 protein in endoplasmic reticulum stress-loaded neurons.

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.

Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons.

The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

Characterization of guanine and guanosine transport in primary cultured rat cortical astrocytes and neurons.

In this study, we examined the transport mechanisms for guanine and guanosine in rat neurons and astrocytes, and compared their characteristics. In the both types of cell, the uptake of [(3)H]guanine and [(3)H]guanosine was time-, temperature-, and concentration-dependent, and Na(+)-independent. Their uptake decreased on the addition of purine and pyrimidine nucleobases or nucleosides, and the inhibitory effect of the purine analogues was greater than that of the pyrimidine ones. In both cell types, equilibrative nucleoside transporter (ENT) 1 and ENT2 expression was confirmed at the mRNA level, and nitrobenzylmercaptopurine riboside, a representative inhibitor for ENT, decreased their uptake at concentrations of over 10 microM. Comparing uptake characteristics between the substrates, [(3)H]guanine uptake exhibited higher affinity and clearance than [(3)H]guanosine uptake in each type of cell. Although between neurons and astrocytes, there was no difference in the apparent uptake clearance for [(3)H]guanine and [(3)H]guanosine, which was calculated based upon the cellular protein content, the cellular uptake clearance was significantly greater in astrocytes than in neurons. These findings indicate that guanine and guanosine, of which the former is a preferable substrate, are taken up into both neurons and astrocytes via ENT2, and that the extracellular concentrations of guanine and guanosine are mainly regulated by astrocytes to maintain brain physiology.

Mouse equilibrative nucleoside transporter 2 (mENT2) transports nucleosides and purine nucleobases differing from human and rat ENT2.

Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The K(m) values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 microM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.

Anticancer nucleobase analogues 6-mercaptopurine and 6-thioguanine are novel substrates for equilibrative nucleoside transporter 2.

Various antimetabolites of nucleobase analogues, such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 5-fluorouracil (5-FU), are used for cancer treatments. The first step in nucleobase analogue drug therapy is entry of these compounds into tumor cells. Equilibrative nucleoside transporter 2 (ENT2) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we investigated whether or not these nucleobase analogues are transported via ENT2, using mouse ENT2-overexpressing Cos-7 cells. The hypoxanthine uptake mediated by ENT2 was significantly reduced by the addition of 6-MP and 6-TG, and the inhibition of the hypoxanthine uptake by the 6-thiopurines was competitive. Transfection of ENT2 cDNA into Cos-7 cells resulted in an increase in 6-MP uptake. The 6-MP uptake via ENT2 showed clear time- and substrate concentration-dependent profiles, and was inhibited by 6-TG in an inhibitor concentration-dependent fashion. On the other hand, uracil was not a substrate for ENT2, and 5-FU had no effect on the hypoxanthine uptake via ENT2. Therefore, we concluded that 6-MP and 6-TG, but not 5-FU, are transported mediated by the same recognition site on ENT2 with hypoxanthine.

Novel Na+ -independent and adenine-specific transport system for adenine in primary cultured rat cortical neurons.

Endogenous adenine is an important modulator of cell survival and activity in the central nervous system. In the present study, we examined the transport mechanisms for adenine in primary cultured rat cortical neurons and astrocytes. [3H]Adenine was time-dependently taken up into neurons, but not into astrocytes. In kinetic analysis, the [3H]adenine uptake by neurons was observed to be saturable, and an Eadie-Hofstee plot showed that a single component was involved in the uptake, with kinetic parameters of Km=6.09 microM and Vmax=0.340 nmol/mg protein per min. In inhibition assaying by nucleobases and nucleosides, and inhibitors for equilibrative nucleoside transporters, organic ion transporters and peptide transporters, which were reported to transport nucleobases and their analogues, the [3H]adenine uptake by neurons was found to be significantly inhibited by excess concentrations of adenine, hypoxanthine and adenosine, and was greatly reduced only by the addition of adenine. Therefore, it was indicated that adenine in the extracellular fluid in the central nervous system is taken up into neurons, but not into astrocytes, and that neurons may present a novel Na+ -independent and adenine-specific transport system.

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2.

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.

Transport and toxic mechanism for aluminum citrate in human neuroblastoma SH-SY5Y cells.

Aluminum (Al) is thought to be a risk factor for neurodegenerative disorders, but the molecular mechanism has been not clarified yet. In this study, we examined how a transport system handled transport of Al citrate, the major Al species in brain, and effect of Al citrate treatment on expression of the transporter and on susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells. Uptake of Al citrate by the cells was temperature- and concentration-dependent, and inwardly-directed Na(+)-gradient-independent. Simultaneous application and preloading of L-cystine or L-glutamate inhibited and stimulated, respectively, the Al citrate uptake by SH-SY5Y cells, demonstrating kinetically that Na(+)-independent L-cystine/L-glutamate exchanger, system Xc(-), is involved in its uptake. When the cells were treated with Al citrate, but not citrate, for 2 weeks, but not a day, the expression of the transporter was decreased. Although the cell viability and glutathione content of the cells were not altered by the treatment with Al citrate alone, the number of dead cells among the Al citrate-treated cells increased on exposure to oxidative stress caused by a glucose deprivation/reperfusion treatment. These findings demonstrate that Al citrate is a substrate for system Xc(-), and that chronic treatment with Al citrate causes downregulation of the transporter and increases the vulnerability of the cells to oxidative stress without a direct effect on the viability or GSH content.

Contribution of an unidentified sodium-dependent nucleoside transport system to the uptake and cytotoxicity of anthracycline in mouse M5076 ovarian sarcoma cells.

In the present study, we investigated whether an unidentified system for Na(+)-dependent nucleoside transport is expressed by mouse M5076 ovarian sarcoma cells, besides concentrative nucleoside transporter 2 (CNT2(M)), and is involved in the uptake and cytotoxicity of anthracyclines. In a transport assay involving CNT2(M)-transfectants, CNT2(M) was found to transport [(3)H]cytidine in a Na(+)-dependent manner, and 500 microM cytidine completely inhibited the Na(+)-dependent uptake of [(3)H]uridine via the transporter. In contrast, the Na(+)-dependent [(3)H]uridine uptake by M5076 cells decreased with 500 microM cytidine only to 70% of the control level. Furthermore, transfection of CNT2(M)-specific siRNAs into M5076 cells resulted in a reduction in the Na(+)-dependent uptake of [(3)H]uridine by only 23%, although the expression of CNT2(M) mRNA and Na(+)-dependent uptake of [(3)H]cytidine disappeared in the cells. The uptake of pirarubicin (THP), an anthracycline, by M5076 cells requiring extracellular Na(+) was significantly inhibited by 500 microM uridine, but not 500 microM cytidine. The Na(+)-dependent and cytidine-insensitive uptake of [(3)H]uridine and the that of THP by M5076 cells significantly increased on cotreatment with both cholate and taurocholate, and the enhancement of THP uptake by the bile acids was reversed by cotreatment with 500 microM uridine. Furthermore, the cytotoxicity of THP and doxorubicin, which were previously reported to be taken up via the same transporter, toward M5076 cells was enhanced by cotreatment with both the bile acids. Therefore, it was indicated that an unidentified Na(+)-dependent transport system for nucleosides is expressed by M5076 cells, and contributes to the uptake and cytotoxicity of the anthracyclines.

Transport mechanisms for adenosine and uridine in primary-cultured rat cortical neurons and astrocytes.

Endogenous adenosine and uridine are important modulators of neural survival and activity. In the present study, we examined transport mechanisms of adenosine and uridine in primary-cultured rat cortical neurons, and compared the results for neurons with those for astrocytes. Reverse transcription-polymerase chain reaction identified the mRNAs for ENT1, ENT2, and CNT2, but not CNT1 and CNT3, in neurons and astrocytes. [3H]Adenosine and [3H]uridine were time-, temperature-, and concentration-dependently taken up into neurons and astrocytes. In kinetic analyses, the uptake of both substrates by neurons and astrocytes consisted of two and one, respectively, saturable transport components. The uptake clearance for both substrates by neurons was greater than that by astrocytes. The relative contribution of the high-affinity major component of both substrates to total uptake was estimated to be approximately 80% in neurons. The uptake of [3H]adenosine and [3H]uridine by both neurons and astrocytes was almost entirely Na+-independent, and sensitive to micro, but not nano, molar concentrations of nitrobenzylmercaptopurine riboside, which are transport characteristics of ENT2. Therefore, it was indicated that adenosine and uridine are more efficiently taken up into neurons than into astrocytes, and ENT2 may predominantly contribute to the transport of the nucleosides as a high-affinity transport system in neurons, as in the case of astrocytes.

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D.

Cytochalasin D (CD) induced production of the superoxide radical (O(2)(-)) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O(2)(-) production. CD as well as PMA induced the translocation of p47(phox) to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCzeta antagonist with sequences based on the endogenous PKCzeta pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O(2)(-) production. On the other hand, the production of O(2)(-) induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.

Uptake of the anthracycline pirarubicin into mouse M5076 ovarian sarcoma cells via a sodium-dependent nucleoside transport system.

PURPOSE: We have previously demonstrated that the cytotoxicity of anthracyclines, pirarubicin (THP) and doxorubicin (DOX), is partially dominated by their intracellular amounts, which depend on the uptake efficacy of transporter(s). To clarify their transport mechanism, we examined whether or not Na+/nucleoside cotransporter (CNT) is involved in the uptake of THP by M5076 cells. METHODS: Expression of the CNT isoforms was determined by reverse-transcription PCR. We used two cell lines, intact M5076 and CNT2-transfected Cos-7 cells, to characterize the uptake of THP and [3H]uridine. RESULTS: The mRNA for CNT2, but not that for CNT1 or CNT3, was expressed in M5076 cells, and [3H]uridine uptake by the cells required a Na+ gradient as a driving force. THP uptake by M5076 cells depended on a Na+ gradient, and furthermore, formycin B and AZT had cis-inhibitory and trans-stimulatory effects on the uptake. The efflux of [3H]uridine from M5076 cells was stimulated by the addition of THP extracellularly, which constituted definite evidence of CNT-mediated uptake of THP. However, THP uptake by CNT2 transfectant was almost the same as that by mock cells, indicating that an unidentified CNT isoform contributes to THP uptake by M5076 cells, this being supported by the differences in transport characteristics of [3H]uridine between M5076 and CNT2-transfected cells. CONCLUSION: THP is partially taken up into M5076 cells via a novel Na+-dependent transport system common to nucleosides.

Transport mechanism for aluminum citrate at the blood-brain barrier: kinetic evidence implies involvement of system Xc- in immortalized rat brain endothelial cells.

Although accumulation of aluminum (Al) in the brain is known to cause neurodegenerative disorders and to be regulated mainly by the blood-brain barrier (BBB), the mechanism responsible for Al transport at the BBB has not been clarified yet. In this study, we investigated what kind of transporter is involved in the transport of Al citrate, which is the major species of Al in the brain, at the BBB using a rat immortalized brain endothelial cell line (RBEC1), focusing on the glutamate transporter family. The uptake of Al citrate showed temperature- and concentration-dependency, and did not require an inwardly directed Na+-gradient as a driving force, ruling out the involvement of Na+-dependent glutamate transporters in its transport. By RT-PCR, in RBEC1, there were mRNAs for the components of a Na+-independent glutamate transporter, system Xc-. L-Glutamate and L-cystine, representative ligands for system Xc-, significantly inhibited the uptake of Al citrate, and loading of them into the cells resulted in stimulation of its uptake in RBEC1. These results demonstrated that Al citrate is taken up into RBEC1 via system Xc-, and that this system might play an important role in Al citrate transport at the BBB.