Kazuki Nagasawa

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.

Membrane Transport and Antitumor Activity of Pirarubicin, and Comparison with Those of Doxorubicin

We have compared the membrane transport and antitumor activity of pirarubicin with those of doxorubicin in M5076 ovarian sarcoma, which exhibits low sensitivity to doxorubicin. Pirarubicin was rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reached more than 2.5‐fold that of doxorubicin. In terms of the 50% cell growth‐inhibitory concentration in vitro, pirarubicin was more effective than doxorubicin. Thus, the intracellular concentration influenced the cytotoxicity of these anthracycline agents. On comparison of the nuclear uptake of pirarubicin and doxorubicin, the nucleus/cell ratio of pirarubicin was found to be about 40%, whereas that of doxorubicin reached more than 80%. As the intranuclear concentration of pirarubicin is dependent on nuclear transport, the increases in not only cell membrane transport, but also nuclear membrane transport contributed to the enhancement of the efficacy of pirarubicin. In M5076 solid tumor‐bearing mice, pirarubicin reduced the tumor weight to 60% of the control level, although doxorubicin had no effect. These results were supported by the intracellular uptake of pirarubicin. Moreover, theanine, which inhibited the pirarubicin efflux from M5076 cells, increased by 1.3‐fold the pirarubicin concentration in the tumor and enhanced the therapeutic efficacy of pirarubicin 1.7‐fold. In conclusion, our results suggest that an increase in the concentration of an anthracycline derivative in tumor cells due to alteration of cell membrane transport results in enhancement of the antitumor activity.

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells

Abstract We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks’ balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (Km), maximum velocity (Vmax) and Vmax/Km) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0°C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.

Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

We previously indicated that lovastatin acid, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, was transported by a monocarboxylate transporter (MCT) in cultured rat mesangial cells. In this study, to identify the MCT isoform(s) responsible for the lovastatin acid uptake, the transport mechanism was investigated using bovine kidney NBL-1 cells, which have been reported to express only MCT4 at the protein level. On RT-PCR analysis, the message of mRNAs for MCT1 and MCT4 was detected in the NBL-1 cells used in this study, which was confirmed by kinetic analysis of [14C]L-lactic acid uptake, consisting of high- and low-affinity components corresponding to MCT1 and MCT4, respectively. The lovastatin acid uptake depended on an inwardly directed H+-gradient, and was inhibited by representative monocarboxylates, but not by inhibitors/substrates for organic anion transporting polypeptides and organic anion transporters. In addition, L-lactic acid competitively inhibited the uptake of lovastatin acid and lovastatin acid inhibited the low affinity component of [14C]L-lactic acid uptake dose dependently. The inhibition constant of L-lactic acid for lovastatin acid uptake was almost the same as the Michaelis constant for [14C]L-lactic acid uptake by the low-affinity component. These kinetic evidences imply that lovastatin acid was taken up into NBL-1 cells via MCT4.

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.

Contribution of Specific Transport Systems to Anthracycline Transport in Tumor and Normal Cells

Anthracycline antibiotics are very effective neoplastic agents widely used clinically. However, because of their many adverse effects (e.g. cardiotoxicity, leukopenia and alopecia), their clinical use has been limited. In order to minimize their adverse effects in clinical cancer chemotherapy, anthracyclines must be selectively transported into tumor cells. If there are differences in transport characteristics between tumor and normal cells, it should be possible to establish a strategy for selectively delivering anthracyclines to tumor cells on the basis of the differences. In human cultured leukemia HL60 cells, as tumor cells, and human fresh mononuclear cells, as normal cells, doxorubicin, pirarubicin, daunorubicin and idarubicin were incorporated via a common carrier-mediated system, but the carriers were different in the two cell types. In HL60 cells, it was indicated that a nucleoside transport system contributed, at least in part, to the transport of doxorubicin and pirarubicin, but not daunorubicin and idarubicin, and its contribution to pirarubicin transport was found in other tumor cells, i.e. mouse ovarian sarcoma M5076 and Ehrlich ascites carcinoma cells. On the other hand, in mononuclear cells, there was no involvement of a nucleoside transport system for the four anthracyclines examined. Therefore, we thought that with the modification of an anthracycline molecule as a substrate for the nucleoside transport system, the anthracycline could be delivered selectively to tumor cells.

Transport mechanisms of idarubicin, an anthracycline derivative, in human leukemia HL60 cells and mononuclear cells, and comparison with those of its analogs

Transport mechanisms of idaruhicin (IDA) in HL60 cells, as leukemia cells, and human mononuclear cells (MNCs), as normal cells, were investigated, and compared with those of its analogs. The uptake of IDA by both cell types was temperature‐ and concentration‐dependent, was inhibited competitively by daunorubicin (DNR) and noncompetitively by adriamycin (ADR), and was stimulated by preloading of the cells with DNR and ADR, indicating the partial involvement of a carrier‐mediated mechanism. On pretreatment of the cells with 2,4‐dinitrophenol, IDA uptake by HL60 cells increased, but that by MNCs decreased, suggesting that IDA was partially taken up into HL60 cells via an energy‐independent carrier system, and into MNCs via an energy‐dependent one. We speculated that in HL60 cells the carrier concerned with IDA uptake was common to DNR and ADR, and that the binding site of IDA on the carrier was the same as that for DNR, but not that for ADR, while in MNCs the carrier system consisted of, at least in part, a carrier for DNR uptake and one for ADR uptake, and the binding site of IDA was identical to that for DNR in the former, but different from that for ADR in the latter. It appeared that the uptake of IDA was greater than those of pirarubicin, DNR and ADR in both HL60 cells and MNCs, and that IDA was incorporated into MNCs more efficiently than into HL60 cells because of the higher uptake efficacy of the carrier(s).

Inhibitory effect of statins on fetal bovine serum‐induced proliferation of rat cultured mesangial cells and correlation between their inhibitory effect and transport characteristics

Mesangial cells play an important role in physiologic functions, including the regulation of glomerular filtration, and as a pathogenic factor for proliferative glomerulonephritis. We compared the potencies of the inhibitory effects of simvastatin acid, lovastatin acid, and pravastatin on fetal bovine serum (FBS)-induced proliferation of rat cultured mesangial cells, and examined the correlation between their inhibitory effects and intracellular concentrations. We also investigated the transport of the statins in the cells, and whether or not their intracellular concentrations were determined by their transport characteristics. It appeared that the growth inhibitory effects on FBS-induced proliferation of mesangial cells of simvastatin acid and lovastatin acid were approximately the same, but that of pravastatin was extremely weak compared with the others. The growth inhibitory effects of these agents were suggested to depend, at least in part, on the amount incorporated intracellularly. Simvastatin acid, lovastatin acid, and pravastatin appeared to be taken up by mesangial cells via a common carrier, the uptake capacity being determined by their lipophilicity. Therefore, it was thought that the growth inhibitory effects of the statins partially depended on their carrier-mediated uptake by mesangial cells.