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DOI： 10.1016/j.ceca.2021.102386

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The electrogenicity of mitochondrial Na+-Ca2+ exchange (NCXm) had been controversial and no membrane current through it had been reported. We succeeded for the first time in recording NCXm-mediated currents using mitoplasts derived from mouse ventricle. Under conditions that K+, Cl-, and Ca2+ uniporter currents were inhibited, extra-mitochondrial Na+ induced inward currents with 1 μM Ca2+ in the pipette. The half-maximum concentration of Na+ was 35.6 mM. The inward current was diminished without Ca2+ in the pipette, and was augmented with 10 μM Ca2+. The Na+-induced inward currents were largely inhibited by CGP-37157, an NCXm blocker. However, the reverse mode of NCXm, which should be detected as an outward current, was hardly induced by extra-mitochondrial application of Ca2+ with Na+ in the pipette. It was concluded that NCXm is electrogenic. This property may be advantageous for facilitating Ca2+ extrusion from mitochondria, which has large negative membrane potential.

DOI： 10.1186/s12576-020-00752-3

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It has been an unsolved question how cardiac mitochondrial energetics is regulated during working transition. Mathematical modelling is a powerful tool to explore the complicated networks of mitochondrial metabolism. We summarise recent progress and remaining questions about mitochondrial energetics in heart, especially focusing on approaches utilizing mathematical modelling. Feedback activation by ADP and/or inorganic phosphate is an old, but still an attractive hypothesis for explaining regulation mechanisms of cardiac mitochondrial energetics. However, this hypothesis has not been fully validated by experiments, because rises of ADP and/or inorganic phosphate concentrations during cardiac workload increase have not been detected in many experiments. The hypothesis of intracellular energetic units is an extended version of feedback activation, which has a similar problem. The each-step activation hypothesis beautifully reproduces metabolites constancy, although such master regulators have not been identified yet. Ca2+ has been the most plausible candidate because some of the mitochondrial dehydrogenases are activated by Ca2+ . Recent experimental and simulation studies, however, throw doubt on its physiological relevance. Finally we discuss issues to be solved to obtain a better view of cardiac mitochondrial energetics. This article is protected by copyright. All rights reserved.

DOI： 10.1113/JP276817

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Roles of mitochondrial Na+-Ca2+ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca2+ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca2+ recycling via NCLX and sarco/endoplasmic reticulum Ca2+-ATPase SERCA, and interaction with store-operated Ca2+ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.

DOI： 10.1016/j.ceca.2019.102114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Aims Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4.
Methods and results Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent K-d = similar to 500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+-and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (>= six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced.
Conclusions These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

DOI： 10.1093/cvr/cvx117

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca2+, because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca2+-dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca2+ and inorganic phosphate (Pi) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca2+-dependent activations of the aspartate/glutamate carrier and the F1Fo-ATPase, and the Pi-dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic-Ca2+ concentration ([Ca2+](cyt)) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2-oxoglutaratewere used asmitochondrial substrates, the energymetabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca2+](cyt). It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiacworkload transition, and Ca2+ has only a minor role under physiological conditions.

DOI： 10.1113/JP272598

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DOI： 10.1016/j.bbamcr.2016.08.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Introduction Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies.
Method and Results
We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed after depolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 mu M S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05).
Conclusions
We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.

DOI： 10.1371/journal.pone.0164795

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na+-dependent mitochondrial Ca2+ efflux transporter, NCLX, regulates B lymphocyte chemotaxis. Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca2+ was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes. Chelating intracellular Ca2+ with BAPTA-AM disturbed CXCL12-induced chemotaxis, suggesting that modulation of cytosolic Ca2+ via NCLX, and thereby Rac1 activation and F-actin polymerization, is essential for B lymphocyte motility and chemotaxis. Mitochondrial polarization, which is necessary for directional movement, was unaltered in NCLX-knockdown cells, although CXCL12 application failed to induce enhancement of mitochondrial polarization, in contrast to control cells. Mouse spleen B lymphocytes were similar to the cell lines, in that pharmacological inhibition of NCLX by CGP-37157 diminished CXCL12-induced chemotaxis. Unexpectedly, spleen T lymphocyte chemotaxis was unaffected by CGP-37157 treatment, indicating that NCLX-mediated regulation of chemotaxis is B lymphocyte-specific, and mitochondria-endoplasmic reticulum Ca2+ dynamics are more important in B lymphocytes than in T lymphocytes. We conclude that NCLX is pivotal for B lymphocyte motility and chemotaxis.

DOI： 10.1038/srep28378

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.5360/membrane.41.215

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Other Link： http://search.jamas.or.jp/link/ui/2017017032

Language：English   Publishing type：Research paper (scientific journal)   Publisher：KOREAN PHYSICAL SOC  

The antigen binding to the B cell receptor (BCR) induces a rise in cytoplasmic Ca2+ and initiates cell responses such as apoptosis, proliferation, and differentiation in B lymphocytes. We previously showed that the inhibition of mitochondrial Na+-Ca2+ exchange (NCXm) by 7-chloro-5- (2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157] dose-dependently reduced BCR-mediated rise in cytoplasmic Ca2+ in DT40 B lymphocytes. However, the effect of CGP-37157 on BCR-mediated apoptosis is not yet fully understood. Here, we studied the effect of NCXm inhibition on BCR-mediated apoptosis in DT40 B lymphocytes. Apoptotic cells (Annexin V+ and PI - ) were increased by the BCR-stimulation while the treatment of cells with 20-mu M CGP-37157 attenuated the apoptosis induction. On the other hand, 24-hr treatments with 0.2 - 20 mu M CGP- 37157 without the BCR stimulation did not affect the apoptotic-cell population. In the NCLX (a gene encoding NCXm) knockout cells, although the apoptotic cells were increased in the control, the appearance of late apoptotic cells (Annexin V+ and PI+) was decreased by BCR stimulation when compared to the wild-type cells. Our findings suggest that the inhibition of NCXm attenuates BCR-mediated apoptosis in DT40 B lymphocytes.

DOI： 10.3938/jkps.67.1915

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：SPRINGER JAPAN KK  

Mitochondrial Ca2+ is known to regulate diverse cellular functions, for example energy production and cell death, by modulating mitochondrial dehydrogenases, inducing production of reactive oxygen species, and opening mitochondrial permeability transition pores. In addition to the action of Ca2+ within mitochondria, Ca2+ released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca2+ dynamics have been identified: a mitochondrial Ca2+ uniporter (MCU), a mitochondrial Na+-Ca2+ exchanger (NCLX), and a candidate for a mitochondrial H+-Ca2+ exchanger (Letm1). In this review, we focus on the mitochondrial Ca2+ release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca2+ release system is not only crucial in maintaining mitochondrial Ca2+ homeostasis but also participates in the Ca2+ crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.

DOI： 10.1007/s12576-014-0326-7

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.5105/jse.34.69

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Background-The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provides therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells. The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. Methods and Results-To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse pluripotent stem cell-derived cardiomyocytes and identified 4 chemical compound groups: inhibitors of glycogen synthase kinase-3, p38 mitogen-activated protein kinase, and Ca 2+/calmodulin-dependent protein kinase II, and activators of extracellular signal-regulated kinase. Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human pluripotent stem cells, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas extracellular signal-regulated kinase activators and Ca2+/calmodulin-dependent protein kinase II inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, glycogen synthase kinase-3 and p38 mitogen-activated protein kinase inhibitors substantially and synergistically induced re-entry and progression of cell cycle in neonatal but also as well as adult cardiomyocytes. Conclusions-Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered pluripotent stem cell-derived cardiomyocytes as a potent tool to explore chemical-based cardiac regenerative strategies. © 2013 American Heart Association, Inc.

DOI： 10.1161/CIRCGENETICS.113.000330

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Mitochondrial Ca2+ is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na+-Ca2+ exchanger (NCXmit), a Ca2+ extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca2+ oscillation and action potential generation. The upstrokes of action potential and Ca2+ transient were markedly slower, and sarcoplasmic reticulum (SR) Ca2+ handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca2+ content to slow spontaneous SR Ca2+ leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca2+ handling.

DOI： 10.1038/srep02766

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Language：English   Publishing type：Research paper (international conference proceedings)  

Mitochondria Na+-Ca2+ exchange (NCXmit) was first discovered by Carafoli et al. in 1974. Thereafter, the mechanisms and roles of NCXmit have been extensively studied. We review NCX mit in cardiomyocytes and lymphocytes by presenting our recent studies on it. Studies of NCXmit in rat ventricular cells demonstrated that NCXmit is voltage dependent and electrogenic. A targeted knockdown and knockout of NCLX in HL-1 cardiomyocytes and B lymphocytes, respectively, significantly reduced the NCXmit activity, indicating that NCLX is a major component of NCXmit in these cells. The store-operated Ca2+ entry was greatly attenuated in NCLX knockout lymphocytes, suggesting that substantial amount of Ca2+ enters into mitochondria and is released to cytosol via NCXmit. NCXmit or NCLX has pivotal roles in Ca2+ handling in mitochondria and cytoplasm. © Springer Science+Business Media New York 2013.

DOI： 10.1007/978-1-4614-4756-6-16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca2+ handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na+/Ca2+ exchanger-1 (NCX-1) activity. Na+/H+ exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca2+ handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca2+ transient in ISO group was normalized in ISO + BIIB723 group. The significant increase in amplitude of multi-cellular Ca2+ waves (CaW) generated at high [Ca2+](o) of LV slices in ISO group was also normalized in ISO + BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca2+ handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca2+ transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca2+ transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.02.041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Cytoplasmic Ca2+ concentration ([Ca2+](i)) increases upon activation of antigen-receptor in lymphocytes. Mitochondria have been suggested to regulate the [Ca2+](i) response, but the molecular mechanisms and the roles are poorly understood. To clarify them, we carried out a combination study of mathematical simulations and knockout or knockdown of NCLX, a gene candidate for themitochondrial Na+-Ca2+ exchanger (NCXmit), in B lymphocytes. A mathematical model of Ca2+ dynamics in B lymphocytes demonstrated that NCXmit inhibition reduces basal Ca2+ content of endoplasmic reticulum (ER) and suppresses B-cell antigen receptor (BCR)-mediated [Ca2+](i) rise. The predictions were validated in DT40 B lymphocytes of heterozygous NCLX knockout (NCLX+/-). In NCLX+/- cells, mitochondrial Ca2+ efflux via NCXmit was strongly decelerated, suggesting NCLX is a gene responsible for NCXmit in B lymphocytes. Consistent with the predictions, ER Ca2+ content declined and [Ca2+](i) hardly rose upon BCR activation in NCLX+/- cells. ER Ca2+ uptake was reduced to similar to 58% of the wild-type (WT), while it was comparable toWT whenmitochondrial respiration was disturbed. Essentially the same results were obtained by a pharmacological inhibition or knockdown of NCLX by siRNA in A20 B lymphocytes. Unexpectedly, ER Ca2+ leak was augmented and co-localization of mitochondria with ER was lower in NCLX+/- and NCLX silenced cells. Taken together, we concluded that NCLX is a key Ca2+ provider to ER, and that NCLX-mediated Ca2+ recycling between mitochondria and ER is pivotal in B cell responses to antigen.

DOI： 10.1113/jphysiol.2011.222927

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K(v)1.5, Na(v)1.5, Hcn4, Ca(v)3.2, Ca(v)1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca2+ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca2+ transients were recapitulated by a simulation model. Although a Ca2+ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca2+ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2011.09.006

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Rationale: Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.
Method and Result: We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10x10(5) VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.
Conclusion: We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.

DOI： 10.1371/journal.pone.0023657

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.248

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Other Link： https://jlc.jst.go.jp/DN/JALC/00356578680?from=CiNii

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：LANDES BIOSCIENCE  

The Na(+), K(+)-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na(+) ions for extracellular K(+) ions by using ATP-mediated phosphorylation, followed by autodephos-phorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na(+), K(+)-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na(+) current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na(+), K(+)-ATPase based on the structure of the related sarcoplasmic- and endoplasmic-reticulum (SERCA) Ca(2+)-ATPase in a BeF(3)-trapped state, 1,2 in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4 and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 that forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by the Na(+), K(+)-ATPase and by other P-type pumps like the Ca(2+)- and H(+), K(+)-ATPases.

DOI： 10.1038/nature07350

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

The goal of our study was to evaluate the origin of the increased O-2 consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O-2 consumption per minute (mVO(2)) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO(2), i.e., mVO(2) without electrical stimulation, was significantly smaller, but mVO(2) for the total Ca2+ handling in excitation-contraction coupling (E-C coupling mVO(2)), i.e., delta mVO(2) (=mVO(2) with stimulation - mVO(2) without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E-C coupling mVO(2) was markedly altered in the hypertrophied heart. Namely, mVO(2) consumed by sarcoplasmic reticulum Ca2+-ATPase (SERCA2) was depressed by 40%; mVO(2) consumed by the Na+/K+-ATPase (NKA)-Na+/Ca2+ exchange (NCX) coupling was increased by 100%. The depressed mVO(2) consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser(16) phospholamban and SERCA2. The increase in NKA-NCX coupling mVO(2) was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E-C coupling mVO(2) in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca(2+)) and increased NCX activity coupled to NKA activity (1ATP: Ca2+). Taken together, we conclude that the energetically less efficient Ca2+ extrusion pathway evenly contributes to Ca2+ handling in E-C coupling in the present hypertrophy model.

DOI： 10.1007/s12576-008-0006-6

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Language：English   Publisher：ROYAL SOC  

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na(+), K(+)-ATPase pump, this alternating gating effects outward transport of three Na(+) ions and inward transport of two K(+) ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed. Binding of the marine toxin, palytoxin, to the Na(+), K(+)-ATPase uncouples the two gates, so that although each gate still responds to its physiological ligand they are no longer constrained to open and close alternately, and the Na(+), K(+)-ATPase is transformed into a gated cation channel. Millions of Na(+) or K(+) ions per second flow through such an open pump-channel, permitting assay of single molecules and allowing unprecedented access to the ion transport pathway through the Na(+), K(+)-ATPase. Use of variously charged small hydrophilic thiol-specific reagents to probe cysteine targets introduced throughout the pump's transmembrane segments allows mapping and characterization of the route traversed by transported ions.

DOI： 10.1098/rstb.2008.0243

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P-type ATPases pump ions across membranes, generating steep electrochemical gradients that are essential for the function of all cells. Access to the ion-binding sites within the pumps alternates between the two sides of the membrane(1) to avoid the dissipation of the gradients that would occur during simultaneous access. In Na(+),K(+)-ATPase pumps treated with the marine agent palytoxin, this strict alternation is disrupted and binding sites are sometimes simultaneously accessible from both sides of the membrane, transforming the pumps into ion channels (see, for example, refs 2, 3). Current recordings in these channels can monitor accessibility of introduced cysteine residues to water-soluble sulphydryl-specific reagents(4). We found previously(5) that Na(+),K(+) pump-channels open to the extracellular surface through a deep and wide vestibule that emanates from a narrower pathway between transmembrane helices 4 and 6 (TM4 and TM6). Here we report that cysteine scans from TM1 to TM6 reveal a single unbroken cation pathway that traverses palytoxin-bound Na(+),K(+) pump-channels from one side of the membrane to the other. This pathway comprises residues from TM1, TM2, TM4 and TM6, passes through ion-binding site II,and is probably conserved in structurally and evolutionarily related P-type pumps, such as sarcoplasmic- and endoplasmic-reticulum Ca(2+)-ATPases and H(+),K(+)-ATPases.

DOI： 10.1038/nature07350

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

To quantitatively understand intracellular Na+ and Cl- homeostasis as well as roles of Na+/K+ pump and cystic fibrosis transmembrane conductance regulator Cl- channel (I-CFTR) during the PI-adrenergic stimulation in cardiac myocyte, we constructed a computer model of beta 1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na+, K+, Ca2+ and Cl-), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca2+ transient and cell shortening, and the decreases in both Cl- concentration and cell volume. These results are consistent with experimental data. Increasing the density Of ICFTR shortened APD and augmented the peak amplitudes of the L-type Ca2+ current (I-CaL) and the Ca2+ transient during the beta 1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I-CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na+ during the beta-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na+ affinity of Na+/K+ pump by protein kinase A. However it was predicted that Na+ increases at higher beating rate because of larger Na+ influx through forward Na+/Ca2+ exchange. It was demonstrated that dynamic changes in Na+ and Cl- fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes. (C) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pbiomolbio.2007.07.005

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Na+/K+ pump is one of key mechanisms to maintain cell volume. When it is inhibited, cells are at risk of swelling. However, in guinea pig ventricular myocytes, the cell area as an index of cell volume was almost constant during 90 min Na+/K+ pump blockade with 40 mu M ouabain despite the marked membrane depolarization. In this study, involvements of Ca2+ transporters and channels in the cardiac cell volume regulation were proposed by conducting the computer simulation in parallel with the experimental validation.

DOI： 10.1196/annals.1387.020

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Although the Na+/K+ pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl- and water fluxes) predicted roles for the plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger, in addition to low membrane permeabilities for Na+ and Cl-, in maintaining cell volume. PMCA might help maintain the [Ca2+] gradient across the membrane though compromised, and thereby promote reverse Na+/Ca2+ exchange stimulated by the increased [Na+](i) as well as the membrane depolarization. Na+ extrusion via Na+/Ca2+ exchange delayed cell swelling during Na+/K+ pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na+/Ca2+ exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl-conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl- efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na+/K+ pump block activated the window current of the L-type Ca2+ current, which increased [Ca2+](i). Finally, the activation of Ca2+-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na+ accompanied by the Cl(-)influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca2+ channels predicted in the simulation was demonstrated in experiments, where blocking Ca2+ channels resulted in a much delayed cell swelling.

DOI： 10.1085/jgp.200609646

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOCIETY  

We aim at introducing a Cl- homeostasis to the cardiac ventricular cell model (Kyoto model), which includes the sarcomere shortening and the mitochondria oxidative phosphorylation. First, we examined mechanisms underlying the cell volume regulation in a simple model consisting of Na+/K+ pump, Na+-K+-2Cl(-) cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator, volume-regulated Cl- channel and background Na+, K+ and Cl- currents. The high intracellular Cl- concentration of approximately 30 mM was achieved by the balance between the secondary active transport via NKCC1 and passive currents. Simulating responses to Na+/K+ pump inhibition revealed the essential role of Na+/K+ pump in maintaining the cellular osmolarity through creating the negative membrane potential, which extrudes Cl- from a cell, confirming the previous model study in the skeletal muscle. In addition, this model well reproduced the experimental data such as the responses to hypotonic shock in the presence or absence of beta-adrenergic stimulation. Finally, the volume regulation via Cl- homeostasis was successfully incorporated to the Kyoto model. The steady state was well established in the comprehensive cell model in respect to both the intracellular ion concentrations and the shape of the action potential, which are all in the physiological range. The source code of the model, which can reproduce every result, is available from http://www.sim-bio.org/.

DOI： 10.1098/rsta.2006.1767

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Background. Several examinations have been performed to identify the genes involved in chronic renal failure using 5/6 nephrectomized rats. Recently, many systematic techniques for examining molecular expression have been developed. They might also be effective in elucidating the molecular mechanism of progressive renal failure. In this study, digital expression profiling was carried out to construct a subtractive mRNA expression database for the 5/6 nephrectomized kidney.
Methods. One thousand clones were randomly sequenced from 5/6 nephrectomized and sham-operated rat kidney cDNA libraries, respectively, and defined by BLAST search. In silico subtractive analysis was performed to search for genes up- or down-regulated in the 5/6 nephrectomized kidney.
Results. The growth factor-related mRNAs and the mRNAs encoding cytoskeletal or membrane proteins were up- regulated, but the transporter-related mRNAs were down-regulated in the 5/6 nephrectomized kidney database. In silico subtraction revealed that 63 mRNAs were increased and 59 were decreased in the 5/6 nephrectomized kidney. To confirm whether the in silico subtractive database reflected the actual expression of mRNA or protein, 12 known genes were examined by Northern blotting or immunoblotting, respectively. The actual expression of the 12 genes was comparable with the results of in silico subtraction. In addition, we successfully isolated five unknown genes, two up- regulated and three down-regulated in the 5/6 nephrectomized kidney.
Conclusion. We constructed a subtractive mRNA expression database for 5/6 nephrectomized kidney, which reflects the actual alterations in mRNA expression after subtotal nephrectomy. This database may be useful for elucidation of the molecular mechanism of progressive renal failure.

DOI： 10.1111/j.1523-1755.2004.00704.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We investigated organic anion and cation transport activity and the expression of several organic ion transporters in hyperuricemic rat kidney. Feeding oxonic acid, an inhibitor of uric acid metabolism, and uric acid for 10 days significantly increased plasma uric acid level. Plasma creatinine and blood urea nitrogen concentrations also increased in hyperuricemic rats, indicating impaired renal function. The accumulation of organic anions, p-ammohippurate (PAH) and methotrexate, and cations, tetraethylammonium (TEA) and cimetidine, into renal slices was markedly decreased, suggesting decreased transport activity for organic anions and cations at the basolateral membrane in the kidney. The expression levels of basolateral organic anion transporters rOAT1 and rOAT3, and organic cation transporter, rOCT2, significantly decreased in hyperuricemic rat kidney as assessed by mRNA and protein levels. In contrast, the expression of rOCT1 was unaltered by hyperuricemia at both mRNA and protein levels. Moreover, the mRNA expression of kidney-specific organic anion transporters, OAT-K1 and OAT-K2, and organic anion transporting polypeptide (oatp) 1, which localize at the brush-border membrane in the kidney, was unchanged in hyperuricemic rats. In conclusion, we showed decreased basolateral organic anion and cation transport activity, accompanied by a specific decrease in rOAT1, rOAT3 and rOCT2 expression in hyperuricemic rat kidney. These phenomena partly contribute to the changed renal disposition of organic anions and cations in hyperuricemia. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S0006-2952(03)00466-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

To identify novel transporters in the kidney, we have constructed an mRNA data base composed of 1000 overall clones by random sequencing of a male rat kidney cDNA library. After a BLAST search, similar to40% of the clones were unknown and/or unannotated and were screened by measuring the uptake of various compounds using Xenopus oocytes. One clone stimulated the uptake of a-methyl-D-glucopyranoside and therefore was termed rat Na+-dependent glucose transporter 1 (rNaGLT1). The rNaGLT1 cDNA (2173 bp) has an open reading frame encoding a 484-amino acid protein, showing <22% homology to known SGLT and GLUT glucose transporters. a-Methyl-D-glucopyranoside uptake by rNaGLT1 cRNA-injected oocytes showed saturability, with an apparent K-m of 3.7 mm and a coupling ratio of 1:1 with Na+. rNa-GLT1 mRNA was expressed predominantly in the kidney upon Northern blot analysis and reverse transcription-PCR. Reverse transcription-PCR in microdissected nephron segments revealed that rNaGLT1 mRNA was primarily localized in the proximal tubules. A clear signal corresponding to rNaGLT1 protein was recognized in the brush-border (but not basolateral) membrane fraction by immunoblot analysis. The rNaGLT1 mRNA level in the kidney was significantly higher than rat SGLT1 and SGLT2 mRNA levels. These findings suggest that rNaGLT1 is a novel Na+-dependent glucose transporter with low substrate affinity that mediates tubular reabsorption of glucose.

DOI： 10.1074/jbc.M212240200

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We have evaluated the functional consequences of genetic variations in human organic cation transporter hOCT1 (SLC22A1). Three coding single nucleotide polymorphisms (cSNPs) resulted in the amino acid changes Pro283Leu, Arg287Gly and Pro341Leu were assessed. Uptake experiments with transient expression system using HEK293 cells revealed that the variants Pro283Leu and Arg287Gly had completely diminished transport activity. The other variant Pro341Leu had a significantly, but not completely, decreased transport activity. Western blot analysis showed that the expression levels of all three variant proteins in the crude membranes of HEK293 cells were comparable to those of wild type hOCT1.Moreover, the expression of variant proteins at the plasma membrane was confirmed by indirect immunouorescence, indicating that these SNPs did not aject the membrane localization of hOCT1. Present results suggest that the amino acid residues Pro283 and Arg287 have a substantial role in substrate ecognition of hOCT1. © 2003, The Japanese Society for the Study of Xenobiotics. All rights reserved.

DOI： 10.2133/dmpk.18.409

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We characterized the interactions of various compounds with OAT-K1 and OAT-K2, kidney-specific organic anion transporters. By using Madin-Darby canine kidney cells stably transfected with OAT-K1 or OAT-K2 cDNA, the antitumor drug methotrexate, the mycotoxin ochratoxin A, endogenous organic anions (thyroid hormones, taurocholic acid, and conjugated steroids), and the antiretroviral drug zidovudine were shown to be substrates for these transporters. Although the apparent Michaelis constant (K-m) values of methotrexate for OAT-K1 and OAT-K2 were 2.1 and 1.8 muM, respectively, 2.5 mM methotrexate inhibited only 20% of the I-125-thyroid hormones uptake via these transporters. In addition, 100 muM methotrexate did not have any effect on [H-3]zidovudine uptake via OAT-K1 or OAT-K2. Similarly, several substrates caused little or no mutual inhibition at concentrations much higher than their K-m values for these transporters. Moreover, intracellular methotrexate trans-stimulated the OAT-K1 - and OAT-K2-mediated uptake of [H-3]folic acid, but not that of other compounds. Organic anion-transporting polypeptide 2 (oatp2), a liver-type homolog of OAT-K1 and OAT-K2, showed similar events. The inhibition constant values of triiodothyronine and taurocholic acid for [H-3]digoxin uptake in oatp2-expressing oocytes, resulted in 50.4 and 1.48 mM, respectively, which were about 9- and 40-fold higher than their K-m values for oatp2, respectively. These findings suggested that several substrates interact with these transporters at different amino acid residue(s). Taken together, these observations suggested that OAT-K1 and OAT-K2 could serve as multispecific transporters, mediating transport of a wide variety of endogenous substances, xenobiotics, and their metabolites in the kidney, presumably via several interaction sites in their molecules.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

Background. High-dose folinic acid is used to accelerate methotrexate elimination to avoid renal toxicity of the drug. The present study was carried out to examine the role of the renal organic anion transporters OAT-K1 and OAT-K2 in the urinary excretion of methotrexate, especially in the methotrexate-folinic acid rescue therapy.
Methods. Madin-Darby canine kidney cells stably expressing OAT-K1 and OAT-K2 were used for the in vitro transport study; 5/6 nephrectomized rats were used to detect changes in mRNA expression levels of OAT-K1 and OAT-K2 and to evaluate methotrexate pharmacokinetics under conditions of renal insufficiency.
Results. Methotrexate efflux mediated by these transporters in stable transfectants was stimulated in the presence of extracellular folic acid and folinic acid, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate. The mRNA expression levels of OAT-K1 and OAT-K2 were markedly diminished after 5/6 nephrectomy, but those of multidrug resistance associated protein 2, which could transport methotrexate, were maintained. Renal clearance of methotrexate was markedly decreased in 5/6 nephrectomized rats compared with that in sham-operated rats. Additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats but not in 5/6 nephrectomized rats.
Conclusions. The decreased expressions of OAT-K1 and OAT-K2 may be attributable to the longer exposure to methotrexate and ineffective folinic acid rescue. In terms of contributing to patient safety, renal clearance of methotrexate, especially folinic acid-stimulated tubular secretion of the drug via these transporters, would be a key factor in methotrexate therapy.

DOI： 10.1046/j.1523-1755.2001.0600031058.x

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society for the Study of Xenobiotics  

Renal organic anion transporters, OAT-K1 and OAT-K2, are expressed specifically in the brushborder membranes of the proximal straight tubules. Both of them transported structurally unrelated compounds such as antitumor drug methotrexate, various endogenous organic anions (thyroid hormones, taurocholic acid and conjugated steroids), and an antiretroviral drug zidovudine, which possesses no typical anionic moiety. In addition, OAT-K1 and OAT-K2 showed bidirectional transport of substrates. Moreover, methotrexate efflux via these transporters was stimulated in the presence of extracellular folic acid-derivatives, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate.<BR>In the state of renal failure caused by 5/6 nephrectomy, the mRNA expression levels of OAT-K1 and OAT -K2 were significantly diminished, and the renal clearance of methotrexate was markedly decreased compared with that in sham-operated rats. Furthermore, additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats, but not in 5/6 nephrectomized rats. These findings suggested that the decreased expressions of OAT-K1 and OAT-K2 were attributed to the longer exposure to methotrexate and invalidated folinic acid rescue in 5/6 nephrectomized rats.<BR> In conclusion, OAT-K1 and OAT-K2 may play important roles in the renal handling of hydrophobic anionic drugs.

DOI： 10.2133/dmpk.16.134

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We examined the pharmacological role of the renal organic anion transporter OAT-K1, which localizes predominantly in the brush-border membranes of proximal straight tubules, in the urinary excretion of methotrexate and the possibility of its contribution to "folinic acid rescue." With Madin-Darby canine kidney (MDCK) cells stably transfected with OAT-K1 cDNA, OAT-K1-mediated methotrexate accumulation was inhibited in the presence of various folio acid derivatives. These derivatives included aminopterin, 5-methyltetrahydrofolic acid, unlabeled methotrexate, folinic acid (citrovorum factor, leucovorin), and folio acid with apparent inhibition constant values of 0.5, 1.2, 1.8, 8.2, and 14.1 mu M, respectively. In contrast, 10 mu M taurocholic acid and sulfobromophthalein did not inhibit OAT-K1-mediated methotrexate accumulation. In addition, methotrexate efflux was stimulated in the presence of inwardly directed gradients of aminopterin, 5-methyltetrahydrofolic acid, unlabeled methotrexate, folinic acid, and folic acid, but not of uric acid, taurocholic acid, and glutathione, indicating that OAT-K1-mediated methotrexate efflux is stimulated by a folic acid derivatives exchange. In conclusion, OAT-K1 was suggested to enhance the apical efflux of highly accumulated methotrexate in tubular epithelial cells and contribute at least in part to folinic acid rescue by exchanging intracellular methotrexate for extracellular folinic acid.

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Language：English   Publisher：The Japanese Society for the Study of Xenobiotics  

We isolated cDNAs coding rat organic anion transporters, OAT-K1 and OAT-K2, expressed specifically in the brush-border membranes of the proximal straight tubules. In this study, we have examined the physiological roles of OAT-K1 and OAT-K2 in the urinary excretion of anionic drugs with the in vitro and in vivo experiments.<BR> Methotrexate efflux via these transporters in the stable transfectants was stimulated in the presence of extracellular organic anions, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate. In the state of renal failure caused by 5/6 nephrectomy, the mRNA expression levels of OAT-K1 and OAT-K2 were significantly diminished In addition, the renal clearance of methotrexate was markedly decreased in 5/6 nephrectomized rats, compared with that in sham-operated rats. Moreover, additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats but not in 5/6 nephrectomized rats. These findings suggested that the decreased expressions of OAT-K1 and OAT-K2 were attributed to the longer exposure to methotrexate and invalidated folinic acid rescue in 5/6 nephrectomized rats.<BR> In conclusion, OAT-K1 and OAT-K2 may play important roles in the renal handling of hydrophobic anionic drugs.

DOI： 10.2133/dmpk.15.supplement_96

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Renal organic anion transporter OAT-K1 was stably transfected in MDCK cells and examined for its transport characteristics and membrane localization. OAT-K1 mediated both uptake and efflux of methotrexate in the apical membranes. Immunoblotting showed that the apparent molecular mass of the expressed OAT-K1 was 50 kDa, which was comparable to that found in the rat renal brush-border membranes. The OAT-K1-mediated methotrexate transport was significantly inhibited in the presence of several organic anions such as folate and sulfobromophthalein. These findings suggest that OAT-K1 mediates bidirectional methotrexate transport across the apical membranes, and may be involved in the renal handling of methotrexate. Copyright (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)01221-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We have isolated a cDNA coding a new organic anion transporter, OAT-K2, expressed specifically in rat kidney. The OAT-K2 cDNA had an open reading frame encoding a 498-amino acid protein (calculated molecular mass of 55 kDa) that shows 91% identity with the rat kidney-specific organic anion transporter, OAT-K1. Reverse transcription-coupled polymerase chain reaction analyses revealed that the OAT-K2 mRNA was expressed predominantly in the proximal convoluted tubules, proximal straight tubules, and cortical collecting ducts. When expressed in Xenopus oocytes, OAT-K2 stimulated the uptake of hydrophobic organic anions, such as taurocholate, methotrexate, folate, and prostaglandin E-2, although its homolog OAT-K1 transported methotrexate and folate, but not taurocholate and prostaglandin E-2. In MDCK cells stably transfected with the OAT-K1 and OAT-K2 cDNAs, each transporter was localized functionally to the apical membranes and showed transport activity similar to that in the oocyte. Moreover, the efflux of preloaded taurocholate was also enhanced across the apical membrane in OAT-K2 transfectant. The taurocholate transport by OAT-K2-expressing cells showed saturability (K-m = 10.3 mu M). Several organic anions, bile acids, cardiac glycosides, and steroids had potent inhibitory effects on the OAT-K2-mediated taurocholate transport in the transfectant. These findings suggest that the OAT-K2 participates in epithelial transport of hydrophobic anionic compounds in the kidney.

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We reported that mitochondrial Na&lt
sup&gt
+&lt
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-Ca&lt
sup&gt
2+&lt
/sup&gt
exchanger NCLX participates in the generation of automaticity via mitochondria-sarcoplasmic reticulum (SR) Ca2+ crosstalk in HL-1 cells, which are originated from mouse atrial myocytes. However there is little information on the contribution of NCLX and mitochondria-SR Ca&lt
sup&gt
2+&lt
/sup&gt
crosstalk to the automaticity of real pacemaker sinoatrial (SA) node cells. In order to clarify this, we incorporated mitochondrial Ca&lt
sup&gt
2+&lt
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dynamics and a subspace between mitochondria and SR into an SA node cell model, in which spontaneous Ca&lt
sup&gt
2+&lt
/sup&gt
release from SR determines the automaticity. NCLX reduction decreased the SR Ca&lt
sup&gt
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content and thereby decelerated the firing rate. In addition, the larger the fraction of mitochondria facing SR became, the larger the contribution of NCLX reduction to the SR Ca&lt
sup&gt
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/sup&gt
content and firing rate became. Taken together, the model analysis suggested that NCLX-mediated mitochondria-SR Ca&lt
sup&gt
2+&lt
/sup&gt
crosstalk plays important roles in modulating the automaticity of SA node cells.

DOI： 10.11239/jsmbe.52.OS-80

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Cardiomyocyte is known to hardly swell during the Na/K pump block, although it functions to maintain cell volume. Previously we analyzed the mechanism using a comprehensive mathematical model of cardiomyocyte, the Kyoto model, and proposed that plasma membrane Ca pump, reverse mode of Na/Ca exchanger (NCX) and window current of L-type Ca channel are the key factors responsible for the cardiac cell volume regulation. In the present study, we validated the hypothesis by conducting new experiments measuring [Na]i (monitored by SBFI), cell area and membrane potential (monitored by di-8-ANEPPS), with pharmacological interventions. When an NCX blocker 1 &mu;M SEA0400 was applied to the cardiomyocyte after 60 min treatment with 40 &mu;M ouabain, [Na]i started to increase, indicating the Na extrusion via reverse mode of NCX under the Na/K pump block condition. In addition, this drug caused a significant cell swelling, confirming the proposed mechanism that NCX in reverse mode prevents the cell swelling. When exposed to 1 &mu;M isoproterenol, cardiomyocytes swelled after a delay, which was predicted to be triggered by L-type Ca channel window current. Additionally, application of an L-type Ca channel blocker 5 &mu;M nifedipine eliminated the swelling almost completely. These experimental findings strongly supported the model prediction that the Ca transporters and channels take key roles in the cardiac cell volume regulation under the condition of Na/K pump block. [J Physiol Sci. 2007;57 Suppl:S80]

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In general, animal cells tend to swell if Na/K pump is blocked. However, the volume of guinea-pig ventricular myocytes remained almost constant during 3 hours under 40 &mu;M ouabain. The Na/K pump block simulation using the Kyoto model showed that the cell swelling is retarded in parallel to a decrease in the background membrane conductances for Cl^&minus; and Na⁺, suggesting very low conductances for these ions in actual ventricular cells. In addition, it is suggested by the simulation that the active Ca²⁺ extrusion by sarcomlemmal Ca²⁺ pump (PMCA) has an important role in the delayed cell swelling. The transmembrane [Ca²⁺] gradient is partially maintained by PMCA, and this makes the Na/Ca exchanger extrudes Na⁺ in reverse mode, compensating for Na/K pump. In the experiment where the Cl^&minus; leak was increased beyond these compensatory mechanisms by applying 5 &mu;M isopreterenol, cell swelling could be induced, but only after a delay of about 55 min after ouabain application. The simulation revealed a gradual membrane depolarization during the delay, and finally it reached a voltage range of continuous opening of a significant fraction of L-type Ca²⁺ channels (window current). A rapid accumulation of Ca²⁺ subsequently activates Ca²⁺-dependent Na⁺ conductance, which finally initiates the rapid cell swelling. [J Physiol Sci. 2006;56 Suppl:S128]

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Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

researchmap

researchmap

researchmap

researchmap