Faculty Profiles - HIASA Miki

Papers - HIASA Miki

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Polyamine release and vesicular polyamine transporter expression in megakaryoblastic cells and platelets. International journal

Mizuki Uehara, Ayaka Fukumoto, Hiroshi Omote, Miki Hiasa

Biochimica et biophysica acta. General subjects 1868 ( 6 ) 130610 - 130610 2024.6

Language：English Publishing type：Research paper (scientific journal)

Polyamines not only play essential roles in cell growth and function of living organisms but are also released into the extracellular space and function as regulators of chemical transduction, although the cells from which they are released and their mode of release are not well understood. The vesicular polyamine transporter (VPAT), encoded by the SLC18B1 is responsible for the vesicular storage of spermine and spermidine, followed by their vesicular release from secretory cells. Focusing on VPAT will help identify polyamine-secreting cells and new polyamine functions. In this study, we investigated the possible involvement of VPAT in vesicular release of polyamines in MEG-01 clonal megakaryoblastic cells and platelets. RT-PCR, western blotting, and immunohistochemistry revealed VPAT expression in MEG-01 cells. MEG-01 cells secreted polyamines upon A23187 stimulation in the presence of Ca2+, which is temperature-dependent and sensitive to bafilomycin A1. A23187-induced polyamine secretion from MEG-01 cells was reduced by treatment with reserpine, VPAT inhibitors, or VPAT RNA interference. Platelets also expressed VPAT, displaying a punctate distribution, and released spermidine upon A23187 and thrombin stimulation. These findings have demonstrated VPAT-mediated vesicular polyamine release from MEG-01 cells, suggesting the presence of similar vesicular polyamine release mechanisms in platelets.

DOI： 10.1016/j.bbagen.2024.130610

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors

Loc Dinh Hoang, Eriko Aoyama, Miki Hiasa, Hiroshi Omote, Satoshi Kubota, Takuo Kuboki, Masaharu Takigawa

International Journal of Molecular Sciences 24 ( 24 ) 17294 - 17294 2023.12

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

DOI： 10.3390/ijms242417294

Vesicular ATP release from hepatocytes plays a role in the progression of nonalcoholic steatohepatitis Reviewed

Keita Tatsushima, Nao Hasuzawa, Lixiang Wang, Miki Hiasa, Shohei Sakamoto, Kenji Ashida, Nobuyuki Sudo, Yoshinori Moriyama, Masatoshi Nomura

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1867 ( 3 ) 166013 - 166013 2021.3

Functional characterization and tissue localization of the facilitative glucose transporter GLUT12 Reviewed

Shunsuke Matsuo, Miki Hiasa, Hiroshi Omote

The Journal of Biochemistry 168 ( 6 ) 611 - 620 2020.12

Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>Facilitative glucose transporters (GLUTs) play crucial roles in glucose utilization and homeostasis. GLUT12 was initially isolated as a novel GLUT4-like transporter involved in insulin-dependent glucose transport. However, tissue distribution and biochemical properties of GLUT12 are not well understood. In this study, we investigated the basic kinetic properties and tissue distribution of GLUT12. Human GLUT12 and GLUT1 were overexpressed and purified using Ni-NTA column chromatography. Reconstituted proteoliposomes showed time-dependent d-glucose transport activity, which was inhibited by phloretin and dehydroascorbate. Dose dependence of glucose transport revealed a KM and Vmax values of 6.4 mM and 1.2 μmol/mg/min, respectively, indicating that GLUT12 is a high-affinity type GLUT. Glucose transport by GLUT12 was inhibited by ATP and glucose-1-phosphate, glucose-6-phosphate and disaccharides (properties similar to those of GLUT1). Indirect immunohistochemistry revealed the distribution of mouse GLUT12 in the apical region of distal tubules and collecting ducts in the kidney and epithelial cells of the jejunum. In addition to these cells, GLUT12 was present in chromaffin cells in the adrenal medulla, the anterior pituitary lobe, as well as the thyroid and pyloric glands. These tissue distributions suggest a unique function of GLUT12, besides that of an insulin-dependent glucose transport.

DOI： 10.1093/jb/mvaa090

Other Link： http://academic.oup.com/jb/article-pdf/168/6/611/35149301/mvaa090.pdf

Anti-Depressant Fluoxetine Reveals its Therapeutic Effect Via Astrocytes. Reviewed International journal

Manao Kinoshita, Yuri Hirayama, Kayoko Fujishita, Keisuke Shibata, Youichi Shinozaki, Eiji Shigetomi, Akiko Takeda, Ha Pham Ngoc Le, Hideaki Hayashi, Miki Hiasa, Yoshinori Moriyama, Kazuhiro Ikenaka, Kenji F Tanaka, Schuichi Koizumi

EBioMedicine 32 72 - 83 2018.6

Language：English Publishing type：Research paper (scientific journal)

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.

DOI： 10.1016/j.ebiom.2018.05.036

Vesicular nucleotide transporter mediates ATP release and migration in neutrophils Reviewed

Yuika Harada, Yuri Kato, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa

Journal of Biological Chemistry 293 ( 10 ) 3770 - 3779 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Biochemistry and Molecular Biology Inc.

Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT/ mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo. These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.

DOI： 10.1074/jbc.M117.810168

Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling Reviewed

Yoshinori Moriyama, Miki Hiasa, Shohei Sakamoto, Hiroshi Omote, Masatoshi Nomura

PURINERGIC SIGNALLING 13 ( 3 ) 387 - 404 2017.9

Language：English Publisher：SPRINGER

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target.

DOI： 10.1007/s11302-017-9568-1

Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain Reviewed

Yuri Kato, Miki Hiasa, Reiko Ichikawa, Nao Hasuzawa, Atsushi Kadowaki, Ken Iwatsuki, Kazuhiro Shima, Yasuo Endo, Yoshiro Kitahara, Tsuyoshi Inoue, Masatoshi Nomura, Hiroshi Omote, Yoshinori Moriyama, Takaaki Miyaji

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 ( 31 ) E6297 - E6305 2017.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl-. A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT-/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

DOI： 10.1073/pnas.1704847114

Function and expression of a splicing variant of vesicular glutamate transporter 1 Reviewed

Satomi Moriyama, Masafumi Iharada, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1859 ( 5 ) 931 - 940 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Vesicular glutamate transporter (VGLUT) is an active transporter responsible for vesicular storage of glutamate in synaptic vesicles and plays an essential role in glutamatergic neurotransmission. VGLUT consists of three isoforms, VGLUT1, VGLUT2, and VGLUT3. The VGLUT1 variant, VGLUTlv, with an additional 75-base pair sequence derived from a second intron between exons 2 and 3, which corresponds to 25 amino acid residues in the 1st loop of VGLUT1, is the only splicing variant among VGLUTs, although whether VGLUTlv protein is actually translated at the protein level remains unknown. In the present study, VGLUTly was expressed in insect cells, solubilized, purified to near homogeneity, and its transport activity was examined. Proteoliposomes containing purified VGLUTlv were shown to accumulate glutamate upon imposition of an inside-positive membrane potential (Delta psi). The Delta psi-driven glutamate uptake activity requires Cl- and its pharmacological profile and kinetics are comparable to those of other VGLUTs. The retinal membrane contained two VGLUT1 moieties with apparent molecular masses of 65 and 57 kDa. VGLUT1v-specific antibodies against an inserted 25-amino acid residue sequence identified a 65-kDa immunoreactive polypeptide. Immunohistochemical analysis indicated that VGLUTly immunoreactivity is present in photoreceptor cells and is associated with synaptic vesicles. VGLUT1v immunoreactivity is also present in pinealocytes, but not in other areas, including the brain. These results indicated that VGLUTly exists in a functional state in rat photosensitive cells and is involved in glutamatergic chemical transmission. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamem.2017.02.002

Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells Reviewed

Tomoya Takeuchi, Yuika Harada, Satomi Moriyama, Kazuyuki Furuta, Satoshi Tanaka, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa

JOURNAL OF BIOLOGICAL CHEMISTRY 292 ( 9 ) 3909 - 3918 2017.3

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

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A(1)-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A(1) and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

DOI： 10.1074/jbc.M116.756197

Urothelial ATP exocytosis: regulation of bladder compliance in the urine storage phase Reviewed

Hiroshi Nakagomi, Mitsuharu Yoshiyama, Tsutomu Mochizuki, Tatsuya Miyamoto, Ryohei Komatsu, Yoshio Imura, Yosuke Morizawa, Miki Hiasa, Takaaki Miyaji, Satoru Kira, Isao Araki, Kayoko Fujishita, Keisuke Shibata, Eiji Shigetomi, Youichi Shinozaki, Reiko Ichikawa, Hisayuki Uneyama, Ken Iwatsuki, Masatoshi Nomura, William C. de Groat, Yoshinori Moriyama, Masayuki Takeda, Schuichi Koizumi

SCIENTIFIC REPORTS 6 29761 2016.7

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

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wildtype male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

DOI： 10.1038/srep29761

Expression of Vesicular Nucleotide Transporter in the Mouse Retina Reviewed

Satomi Moriyama, Miki Hiasa

BIOLOGICAL & PHARMACEUTICAL BULLETIN 39 ( 4 ) 564 - 569 2016.4

Language：English Publishing type：Research paper (scientific journal) Publisher：PHARMACEUTICAL SOC JAPAN

Vesicular nucleotide transporter (VNUT) is a membrane protein that is responsible for vesicular storage and subsequent vesicular release of nucleotides, such as ATP, and plays an essential role in purinergic chemical transmission. In the present study, we investigated whether VNUT is present in the rodent retina to define the site(s) of vesicular ATP release. In the mouse retina, reverse transcription polymerase chain reaction (RT-PCR) and immunological analyses using specific anti-VNUT antibodies indicated that VNUT is expressed as a polypeptide with an apparent molecular mass of 59 kDa. VNUT is widely distributed throughout the inner and outer retinal layers, particularly in the outer segment of photoreceptors, outer plexiform layer, inner plexiform layer, and ganglion cell layer. VNUT is colocalized with vesicular glutamate transporter 1 and synaptophysin in photoreceptor cells, while it is colocalized with vesicular gamma-aminobutyric acid (GABA) transporter in amacrine cells and bipolar cells. VNUT is also present in astrocytes and Muller cells. The retina from VNUT knockout (VNUT-/-) mice showed the loss of VNUT immunoreactivity. The retinal membrane fraction took up radiolabeled ATP in diisothiocyanate stilbene disulfonic acid (DIDS)-, an inhibitor of VNUT, and bafilomycin A1-, a vacuolar adenosine triphosphatase (ATPase) inhibitor, in a sensitive manner, while membranes from VNUT-/- mice showed the loss of DIDS-sensitive ATP uptake. Taken together, these results indicate that functional VNUT is expressed in the rodent retina and suggest that ATP is released from photoreceptor cells, bipolar cells, amacrine cells, and astrocytes as well as Muller cells to initiate purinergic chemical transmission.

DOI： 10.1248/bpb.b15-00872

Involvement of multiple CCN family members in platelets that support regeneration of joint tissues Reviewed

Chikako Hara, Satoshi Kubota, Takashi Nishida, Miki Hiasa, Takako Hattori, Eriko Aoyama, Yoshinori Moriyama, Hiroshi Kamioka, Masaharu Takigawa

MODERN RHEUMATOLOGY 26 ( 6 ) 940 - 949 2016

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

Objectives: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration.
Methods: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells.
Results: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype.
Conclusions: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

DOI： 10.3109/14397595.2016.1155255

Structure, Function, and Drug Interactions of Neurotransmitter Transporters in the Postgenomic Era Reviewed

Hiroshi Omote, Takaaki Miyaji, Miki Hiasa, Narinobu Juge, Yoshinori Moriyama

ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, VOL 56 56 385 - 402 2016

Language：English Publishing type：Part of collection (book) Publisher：ANNUAL REVIEWS

Vesicular neurotransmitter transporters are responsible for the accumulation of neurotransmitters in secretory vesicles and play essential roles in chemical transmission. The SLC17 family contributes to sequestration of anionic neurotransmitters such as glutamate, aspartate, and nucleotides. Identification and subsequent cellular and molecular biological studies of SLC17 transporters unveiled the principles underlying the actions of these transporters. Recent progress in reconstitution methods in combination with postgenomic approaches has advanced studies on neurotransmitter transporters. This review summarizes the molecular properties of SLC17-type transporters and recent findings regarding the novel SLC18 transporter.

DOI： 10.1146/annurev-pharmtox-010814-124816

Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter Reviewed

Natsuko Togawa, Narinobu Juge, Takaaki Miyaji, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 309 ( 2 ) C71 - C80 2015.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Membrane potential (Delta psi)-driven and Cl--dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na+-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Delta psi- and Cl--dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4'-diisothiocyanostilbene- 2,2'-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Delta psi- and Cl--dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.

DOI： 10.1152/ajpcell.00048.2015

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity Reviewed

Shohei Sakamoto, Takaaki Miyaji, Miki Hiasa, Reiko Ichikawa, Akira Uematsu, Ken Iwatsuki, Atsushi Shibata, Hisayuki Uneyama, Ryoichi Takayanagi, Akitsugu Yamamoto, Hiroshi Omote, Masatoshi Nomura, Yoshinori Moriyama

SCIENTIFIC REPORTS 4 6689 2014.10

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

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic beta-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

DOI： 10.1038/srep06689

Identification of a mammalian vesicular polyamine transporter Reviewed

Miki Hiasa, Takaaki Miyaji, Yuka Haruna, Tomoya Takeuchi, Yuika Harada, Sawako Moriyama, Akitsugu Yamamoto, Hiroshi Omote, Yoshinori Moriyama

SCIENTIFIC REPORTS 4 6836 2014.10

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

Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-D-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H+. SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT).

DOI： 10.1038/srep06836

Immunological Identification of Vesicular Nucleotide Transporter in Intestinal L Cells Reviewed

Yuika Harada, Miki Hiasa

BIOLOGICAL & PHARMACEUTICAL BULLETIN 37 ( 7 ) 1090 - 1095 2014.7

Language：English Publishing type：Research paper (scientific journal) Publisher：PHARMACEUTICAL SOC JAPAN

It is well established that vesicular nucleotide transporter (VNUT) is responsible for vesicular storage of nucleotides such as ATP, and that VNUT-expressing cells can secrete nucleotides upon exocytosis, playing an important role in purinergic chemical transmission. In the present study, we show that VNUT is expressed in intestinal L cells. Immunohistochemical evidence indicated that VNUT is present in glucagon-like peptide 1 (GLP-1) containing cells in rat intestine. VNUT immunoreactivity is not co-localized with GLP-1, a marker for secretory granules, and synaptophysin, a marker for synaptic-like microvesicles (SLMVs). Essentially the same results were obtained for GLUTag clonal L cells. Sucrose density gradient analysis confirmed that VNUT is present the light fraction, unlike secretory granules. These results demonstrate that intestinal L cells express VNUT in either the unidentified organelles at light density other than secretory granules and SLMVs or a subpopulation of SLMVs, and suggest that L cells are purinergic in nature and secrete nucleotides independent of GLP-1 secretion.

DOI： 10.1248/bpb.b14-00275

Vesicular Nucleotide Transport: A Brief History and the Vesicular Nucleotide Transporter as a Target for Drug Development Reviewed

Miki Hiasa, Natsuko Togawa, Yoshinori Moriyama

CURRENT PHARMACEUTICAL DESIGN 20 ( 16 ) 2745 - 2749 2014.5

Language：English Publishing type：Research paper (scientific journal) Publisher：BENTHAM SCIENCE PUBL LTD

Neurons and neuroendocrine cells store nucleotides in vesicles and release them upon stimulation, leading to intercellular purinergic signaling. The molecular machinery responsible for the vesicular storage of nucleotides was a long standing enigma, however, recently the transporter involving in the process was identified. This article summarizes the history of vesicular storage of nucleotides and the identification of the vesicular nucleotide transporter (VNUT) responsible for the process. The significance of VNUT as a drug target to control purinergic chemical transmission is also discussed.

DOI： 10.2174/13816128113199990574

Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets Reviewed

Miki Hiasa, Natsuko Togawa, Takaaki Miyaji, Hiroshi Omote, Akitsugu Yamamoto, Yoshinori Moriyama

Physiological Reports 2 ( 6 ) 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：American Physiological Society

Nucleotides are stored in the dense granules of platelets. The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation. However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood. The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized. We hypothesized that the vesicular nucleotide transporter (VNUT) is also involved in the vesicular storage of nucleotides in platelets. In this article, we present three lines of evidence that VNUT is responsible for the vesicular storage of nucleotides in platelets and that vesicular ATP transport is crucial for platelet function, detection and characterization of VNUT activity in platelets isolated from healthy humans and MEG-01 cells, RNA interference experiments on MEG-01 cells, and studies on nucleotide transport and release with a selective inhibitor.

DOI： 10.14814/phy2.12034

Suppression of increased blood glucose levels in mice by Awa-ban tea following oral administration of mono- and disaccharides Reviewed

Miki Hiasa, Megumi Kurokawa, Hiroshi Akita, Masatomo Harada, Kengo Niki, Kana Ohta, Masaki Shoji, Noriko Echigo, Takashi Kuzuhara

Journal of Functional Foods 8 ( 1 ) 188 - 192 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier Ltd

Awa-ban tea is a uniquely flavored, pickled and anaerobically fermented tea found only in the Tokushima prefecture in the Shikoku region of Japan. Diabetes is a major health problem worldwide. Here, we report that Awa-ban tea suppressed the increase in blood glucose levels after oral administration of maltose, sucrose, or glucose in mice (n = 9-54). Awa-ban tea suppressed the area under the curve (AUC) of blood glucose by 72-83%. Thus, we propose that Awa-ban tea can be used as an alternative functional beverage for diabetes prevention. © 2014 Elsevier Ltd.

DOI： 10.1016/j.jff.2014.03.012

Inhibition of MAO-A and stimulation of behavioural activities in mice by the inactive prodrug form of the anti-influenza agent oseltamivir Reviewed

Miki Hiasa, Yumiko Isoda, Yasushi Kishimoto, Kenta Saitoh, Yasuaki Kimura, Motomu Kanai, Masakatsu Shibasaki, Dai Hatakeyama, Yutaka Kirino, Takashi Kuzuhara

British Journal of Pharmacology 169 ( 1 ) 115 - 129 2013.5

Language：English Publishing type：Research paper (scientific journal)

Background and Purpose Oseltamivir is the most widely prescribed anti-influenza medication. However, in rare instances, it has been reported to stimulate behavioural activities in adolescents. The goal of this study was to determine the molecular mechanism responsible for these behavioural activities. Experimental Approach We performed an in vitro assay of MAO-A, the enzyme responsible for neurotransmitter degradation, using either the active form - oseltamivir carboxylate (OC) or the inactive prodrug - oseltamivir ethyl ester (OEE). We also analysed the docking of MAO-A with OEE or OC in silico. Mouse behaviours after OEE or OC administration were monitored using automated video and computer analysis. Key Results OEE, but not OC, competitively and selectively inhibited human MAO-A. The estimated Ki value was comparable with the Km values of native substrates of MAO-A. Docking simulations in silico based on the tertiary structure of MAO-A suggested that OEE could fit into the inner pocket of the enzyme. Behavioural monitoring using automated video analysis further revealed that OEE, not OC, significantly enhanced spontaneous behavioural activities in mice, such as jumping, rearing, sniffing, turning and walking. Conclusions and Implications Our multilevel analyses suggested OEE to be the cause of the side effects associated with oseltamivir and revealed the molecular mechanism underlying the stimulated behaviours induced by oseltamivir in some circumstances. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.

DOI： 10.1111/bph.12102

Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules Reviewed

Juliana I. Sesma, Silvia M. Kreda, Seiko F. Okada, Catharina van Heusden, Lama Moussa, Lisa C. Jones, Wanda K. O'Neal, Natsuko Togawa, Miki Hiasa, Yoshinori Moriyama, Eduardo R. Lazarowski

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 304 ( 10 ) C976 - C984 2013.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+ regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

DOI： 10.1152/ajpcell.00371.2012

Identification and purification of resorcinol, an antioxidant specific to Awa-ban (pickled and anaerobically fermented) tea Reviewed

Miki Hiasa, Megumi Kurokawa, Kana Ohta, Tomoyuki Esumi, Hiroshi Akita, Kengo Niki, Yasuyuki Yagi, Noriko Echigo, Dai Hatakeyama, Takashi Kuzuhara

Food Res Int. 54 ( 1 ) 72 - 80 2013

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Awa-ban tea (Awa, Tokushima prefecture in Japan; ban, "evening" in Japanese) is a type of tea exclusive to the Tokushima area. Awa-ban tea leaves are unique in that they are produced via pickling and anaerobic fermentation. Here, we report the identification and purification of an antioxidant that is specifically present in Awa-ban tea but is absent in green and black tea. The molecule, identified as resorcinol, was found to exhibit radical-scavenging activity in vitro at levels equivalent to those of (-)-epigallocatechin gallate in green tea. Fluorescence microscopy and flow cytometry results further showed that resorcinol decreased the levels of reactive oxygen species in HEK293T cells, human umbilical vein endothelial cells, and Jurkat cells in a concentration-dependent manner. Moreover, the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that treatment with 10 mM resorcinol did not affect cell viability. Our results indicate that Awa-ban tea has properties that are quite distinct from those of green and black tea and is therefore a unique antioxidant beverage. We also suggest that resorcinol is a novel tea-based antioxidant. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodres.2013.05.036

Functional and Anatomical Identification of a Vesicular Transporter Mediating Neuronal ATP Release Reviewed

Max Larsson, Keisuke Sawada, Cecilie Morland, Miki Hiasa, Lasse Ormel, Yoshinori Moriyama, Vidar Gundersen

CEREBRAL CORTEX 22 ( 5 ) 1203 - 1214 2012.5

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

ATP is known to be coreleased with glutamate at certain central synapses. However, the nature of its release is controversial. Here, we demonstrate that ATP release from cultured rat hippocampal neurons is sensitive to RNAi-mediated knockdown of the recently identified vesicular nucleotide transporter (VNUT or SLC17A9). In the intact brain, light microscopy showed particularly strong VNUT immunoreactivity in the cerebellar cortex, the olfactory bulb, and the hippocampus. Using immunoelectron microscopy, we found VNUT immunoreactivity colocalized with synaptic vesicles in excitatory and inhibitory terminals in the hippocampal formation. Moreover, VNUT immunolabeling, unlike that of the vesicular glutamate transporter VGLUT1, was enriched in preterminal axons and present in postsynaptic dendritic spines. Immunoisolation of synaptic vesicles indicated presence of VNUT in a subset of VGLUT1-containing vesicles. Thus, we conclude that VNUT mediates transport of ATP into synaptic vesicles of hippocampal neurons, thereby conferring a purinergic phenotype to these cells.

DOI： 10.1093/cercor/bhr203

Characterization of the human MATE2 proton-coupled polyspecific organic cation exporter Reviewed

Toshinori Komatsu, Miki Hiasa, Takaaki Miyaji, Takuji Kanamoto, Takuya Matsumoto, Masato Otsuka, Yoshinori Moriyama, Hiroshi Omote

INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 43 ( 6 ) 913 - 918 2011.6

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

Human multidrug and toxic compound extrusion 2 (hMATE2) is a kidney-specific isoform of hMATE1, an exporter of toxic organic cations (OCs) of exogenous and endogenous origins at the final excretion step in the kidneys and liver (Otsuka et al., 2005), and contains a splicing variant, MATE2K, that has an exon of hMATE2 deleted (Masuda et al., 2006). In the present study, we characterized the degree of expression and the transport properties of hMATE2. Quantitative PCR analysis with probes specific for hMATE2 indicated the presence of hMATE2 mRNA in the kidneys, which corresponded to 39% of total mRNA encoding both hMATE2 and hMATE2K. hMATE2-specific antibodies immunostained the renal urinary tubules. Upon expression in HEK293 cells, hMATE2 was localized in intracellular vesicular structures, and thus transport activity of tetraethylammonium (TEA), a typical substrate for MATE transporters, by the cells was not detected. The hMATE2 protein was purified and reconstituted into liposomes. An artificially imposed pH gradient (Delta pH) across the proteoliposomal membrane drove the uptake of TEA. Dissipation of Delta pH by ammonium sulfate effectively inhibited the TEA uptake, while that of the membrane potential by valinomycin had little effect. The profiles of cis-inhibition of TEA transport by hMATE2 and hMATE2K are similar to each other. Thus, both hMATE2 and hMATE2K equally operate in the human kidneys to extrude OCs into the urine. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biocel.2011.03.005

Type 1 Sodium-dependent Phosphate Transporter (SLC17A1 Protein) Is a Cl--dependent Urate Exporter Reviewed

Masafumi Iharada, Takaaki Miyaji, Takahiro Fujimoto, Miki Hiasa, Naohiko Anzai, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 285 ( 34 ) 26107 - 26113 2010.8

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

SLC17A1 protein (NPT1) is the first identified member of the SLC17 phosphate transporter family and mediates the transmembrane cotransport of Na+/P-i in oocytes. Although this protein is believed to be a renal polyspecific anion exporter, its transport properties are not well characterized. Here, we show that proteoliposomes containing purified SLC17A1 transport various organic anions such as p-aminohippuric acid and acetylsalicylic acid (aspirin) in an inside positive membrane potential (Delta psi)-dependent manner. We found that NPT1 also transported urate. The uptake characteristics were similar to that of SLC17 members in its Cl- dependence and inhibitor sensitivity. When arginine 138, an essential amino acid residue for members of the SLC17 family such as the vesicular glutamate transporter, was specifically mutated to alanine, the resulting mutant protein was inactive in Delta psi-dependent anion transport. Heterologously expressed and purified human NPT1 carrying the single nucleotide polymorphism mutation that is associated with increased risk of gout in humans exhibited 32% lower urate transport activity compared with the wild type protein. These results strongly suggested that NPT1 is a Cl--dependent polyspecific anion exporter involved in urate excretion under physiological conditions.

DOI： 10.1074/jbc.M110.122721

Vesicular Inhibitory Amino Acid Transporter Is a Cl-/gamma-Aminobutyrate Co-transporter Reviewed

Narinobu Juge, Akiko Muroyama, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 284 ( 50 ) 35073 - 35078 2009.12

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

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of gamma-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Delta psi) (positive inside) but not Delta pH. VIAAT-mediated GABA uptake had an absolute requirement for Cl- and actually accompanied Cl- movement. Kinetic analysis indicated that one GABA molecule and two Cl- equivalents were transported during one transport cycle. VIAAT in which Glu(213) was specifically mutated to alanine completely lost the ability to take up both GABA and Cl-. Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl- transporter that co-transports Cl- with GABA or glycine in a Delta psi dependent manner. It is concluded that Cl- plays an essential role in vesicular storage of GABA and glycine.

DOI： 10.1074/jbc.M109.062414

Identification of the vesicular nucleotide transporter (VNUT) in taste cells Reviewed

Ken Iwatsuki, Reiko Ichikawa, Miki Hiasa, Yoshinori Moriyama, Kunio Torii, Hisayuki Uneyama

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 388 ( 1 ) 1 - 5 2009.10

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

Taste cells are chemosensory epithelial cells that sense distinct taste qualities. It is the type II taste cell that express G-protein coupled receptors to sense either umami, sweet, or bitter compounds. Whereas several reports have suggested involvement of ATP in taste signal transduction, there is a paucity of molecular information about how ATP is stored and being released. The recent discovery of a novel vesicular nucleotide transporter (VNUT) led us to examine whether VNUT exist in the taste tissue where ATP is to be released for taste signal transmission. Here, we report that VNUT is selectively expressed in type II cell but not in type III taste cell. In addition, we show that during taste bud development VNUT expression is always accompanied by the expression of type II taste cell markers. Our results, together with previous studies, strongly suggest that VNUT plays a role in type II taste cell. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.07.069

Expression and function of TETRAN, a new type of membrane transporter Reviewed

Hironori Ushijima, Miki Hiasa, Takushi Namba, Hyun-Jung Hwang, Tatsuya Hoshino, Shinji Mima, Tomofusa Tsuchiya, Yoshinori Moriyama, Tohru Mizushima

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 374 ( 2 ) 325 - 330 2008.9

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

In contrast to transport across basolateral membranes, the mechanism governing transport Of Organic anions across the luminal membranes Of proximal tubules has remained Unclear. We recently found Tet-racycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in Cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. overexpression of TETRAN in cultured cells facilitated the Uptake of organic anions Such as indomethacin (a NSAID) and fluorescein. The results suggest that TETPAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.07.034

Identification of a vesicular aspartate transporter Reviewed

Takaaki Miyaji, Noriko Echigo, Miki Hiasa, Shigenori Senoh, Hiroshi Omote, Yoshinori Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 ( 33 ) 11720 - 11724 2008.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes and is exocytosed and stimulates neighboring cells by binding to specific cell receptors. Although evidence increasingly supports the occurrence of aspartergic neurotransmission, this process is still debated because the mechanism for the vesicular storage of aspartate is unknown. Here, we show that sialin, a lysosomal H+/sialic acid cotransporter, is present in hippocampal synaptic vesicles and pineal SLMVs. RNA interference of sialin expression decreased exocytosis of aspartate and glutamate in pinealocytes. Proteoliposomes containing purified sialin actively accumulated aspartate and glutamate to a similar extent when inside positive membrane potential is imposed as the driving force. Sialin carrying a mutation found in people suffering from Salla disease (R39C) was completely devoid of aspartate and glutamate transport activity, although it retained appreciable H+/sialic acid cotransport activity. These results strongly suggest that sialin possesses dual physiological functions and acts as a vesicular aspartate/glutamate transporter. It is possible that people with Salla disease lose aspartergic (and also the associated glutamatergic) neurotransmission, and this could provide an explanation for why Salla disease causes severe neurological defects.

DOI： 10.1073/pnas.0804015105

A novel variant of mouse MATE-1 H+/organic cation antiporter with a long hydrophobic tall Reviewed

Ayumi Kobara, Miki Hiasa, Takuya Matsumoto, Masato Otsuka, Hiroshi Omote, Yoshinori Moriyama

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 469 ( 2 ) 195 - 199 2008.1

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

Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H+-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2007.10.010

[Identification, structure and function of a drug transporter at the final step of extrusion]. Reviewed

Hiasa M, Omote H, Moriyama Y

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 1 ) 52 - 58 2008.1

Multidrug and toxic compound extrusion (MATE)-type proteins as anchor transporters for the excretion of metabolic waste products and xenobiotics Reviewed

Y. Moriyama, M. Hiasa, T. Matsumoto, H. Omote

XENOBIOTICA 38 ( 7-8 ) 1107 - 1118 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

1. Multidrug and toxic compound extrusion (MATE)-type transporters, which were first identified as a bacterial drug transporter family, are present in almost all prokaryotes and eukaryotes, and are thus one of the mostly conserved transporter families in nature.
2. Recently, a mammalian MATE transporter was shown to be a long hypothesized electroneutral H(+)/organic cation exporter that is responsible for the excretion of metabolic waste products and xenobiotics at renal brush border membranes and bile canaliculi. Plant MATE-type transporters are involved in the detoxification of metals and secondary metabolites such as phenols through their vesicular storage or extrusion at the plasma membrane.
3. Thus, MATE transporters are involved in one of the basic mechanisms that maintain homeostasis through the excretion of metabolic waste products and xenobiotics in nature.

DOI： 10.1080/00498250701883753

Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H+/organic cation exporter Reviewed

Miki Hiasa, Takuya Matsumoto, Toshinori Komatsu, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 293 ( 5 ) C1437 - C1444 2007.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Mammalian multidrug and toxic compound extrusion ( MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H+-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney ( Otsuka et al. Proc Natl Acad Sci USA 102: 17923-17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587-593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H+-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K+. Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H+/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.

DOI： 10.1152/ajpcell.00280.2007

Exocytosis of D-aspartate from INS-1E clonal beta cells Reviewed

Masafumi Iharada, Miki Hiasa, Ayumi Kobara, Yoshinori Moriyama

BIOLOGICAL & PHARMACEUTICAL BULLETIN 30 ( 7 ) 1329 - 1331 2007.7

Language：English Publishing type：Research paper (scientific journal) Publisher：PHARMACEUTICAL SOC JAPAN

D-Aspartate is present in the central nervous system and various endocrine organs, and modulates their neuroendocrine function. In islets of Langerhans, alpha and beta cells contain D-aspartate. Here we show that INS-1E clonal beta cells contain the highest amount Of D-aspartate. Immunohistochemical analysis with specific antibodies against D-aspartate indicated that D-aspartate is co-localized with insulin. Upon the addition of K+, both D-aspartate and insulin are secreted from the cells in a Ca2+-dependent manner. A Ca2+ ionophore, A23187, also triggers the release Of D-aspartate and insulin in the presence of Ca2+. Bafilomycin A, a specific inhibitor of V-ATPase and V-ATPase-linked secondary transport, inhibits the secretion of D-aspartate. These results support the idea that D-aspartate is present in insulin-containing secretory granules and co-secreted with insulin through exocytosis.

DOI： 10.1248/bpb.30.1329

The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations Reviewed

Hiroshi Omote, Miki Hiasa, Takuya Matsumoto, Masato Otsuka, Yoshinori Moriyama

TRENDS IN PHARMACOLOGICAL SCIENCES 27 ( 11 ) 587 - 593 2006.11

Language：English Publisher：ELSEVIER SCIENCE LONDON

Multidrug and toxic compound extrusion (MATE) proteins, comprising the most recently designated family of multidrug transporter proteins, are widely distributed in all kingdoms of living organisms, although their function is far from understood. The bacterial MATE-type transporters that have been characterized function as exporters of cationic drugs, such as norfloxacin and ethidium, through H+ or Na+ exchange. Plant MATE-type transporters are involved in the detoxification of secondary metabolites, including alkaloids. Mammalian MATE-type transporters are responsible for the final step in the excretion of metabolic waste and xenobiotic organic cations in the kidney and liver through electroneutral exchange of H+. Thus, we propose that members of the MATE family are organic cation exporters that excrete metabolic or xenobiotic organic cations from the body.

DOI： 10.1016/j.tips.2006.09.001

Wide variety of locations for rodent MATE1, a transporter protein that mediates the final excretion step for toxic organic cations Reviewed

Miki Hiasa, Takuya Matsumoto, Toshinori Komatsu, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 291 ( 4 ) C678 - C686 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H+-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923-17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H+/ tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, alpha cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H+/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body.

DOI： 10.1152/ajpcell.00090.2006

Immunohistochemical localization of D-aspartate in islets of Langerhans Reviewed

Miki Hiasa, Yoshinori Moriyama

BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 ( 6 ) 1251 - 1253 2006.6

Language：English Publishing type：Research paper (scientific journal) Publisher：PHARMACEUTICAL SOC JAPAN

D-Aspartate is a putative modulator of neuroendocrine functions, and is present in various neuroendocrine cells as well as the central nervous system. Here we show that the islet of Langerhans is a D-aspartate-containing endocrine organ. Immunohistochemical analysis with specific antibodies against D-aspartate indicated that D-aspartate is present in all islet cells, and predominantly present in a cells and a subpopulation of F-cells. Since these cells are glutamatergic in nature, it is possible that D-aspartate is involved in the glutamate signaling pathways in the islets.

DOI： 10.1248/bpb.29.1251

Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells Reviewed

S Uehara, SK Jung, R Morimoto, S Arioka, T Miyaji, N Juge, M Hiasa, K Shimizu, A Ishimura, M Otsuka, A Yamamoto, P Maechler, Y Moriyama

JOURNAL OF NEUROCHEMISTRY 96 ( 2 ) 550 - 560 2006.1

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

Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage of (L)-glutamate, and plays an essential role in glutamate-mediated intercellular signal transmission in the CNS and in some neuroendocrine cells. Intestinal L cells are the glucose-responsive neuroendocrine cells responsible for the secretion of glucagon-like peptide 1 (GLP-1). We have shown that intestinal L cells express VGLUT2, a VGLUT isoform, which suggests that L cells secrete (L)-glutamate. In the present study, we investigated this possibility using GLUTag mouse clonal L cells. RT-PCR and northern blot analyses revealed expression of the VGLUT1 and VGLUT2 genes, but not of the VGLUT3 gene. Western blot analysis revealed immunological counterparts for VGLUT2, whereas an immunological counterpart of VGLUT1 was not detected. Indirect immunofluorescence microscopy revealed a punctate distribution of VGLUT2 immunoreactivity throughout the cells, which co-localized with GLP-1. Double-labeling immunoelectronmicroscopy confirmed the association of VGLUT2 with GLP-1-containing secretory granules. The membrane fraction exhibited ATP-dependent (L)-glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive to (D,L)-aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts of (L)-glutamate and GLP-1. (D)-Glucose and methyl-alpha-(D)-glucopyranoside, stimulators of exocytosis of GLP-1, also triggered the secretion of (L)-glutamate. The (L)-glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells stored (L)-glutamate in secretory granules and secreted it with GLP-1 by exocytosis. As GLUTag cells and intestinal L cells express kainate receptors and plasma membrane glutamate transporters, these results support the concept of (L)-glutamate-mediated intercellular signaling in the vicinity of intestinal L cells.

DOI： 10.1111/j.1471-4159.2005.03575.x

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