American Society for Biochemistry and Molecular BiologyActa Medica Okayama0021-925829292017Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells39093918ENTomoyaTakeuchiDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYuikaHaradaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatomiMoriyamaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKazuyukiFuruta Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesSatoshiTanaka Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesAdvanced Science Research Center, Okayama UniversityHiroshiOmoteDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesYoshinoriMoriyamaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesMikiHiasaDepartment of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, hiasa@okayama-u.ac.jp. 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 A1-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 A1 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.No potential conflict of interest relevant to this article was reported.Nature Publishing GroupActa Medica Okayama2041-172362015AtPHT4;4 is a chloroplast-localized ascorbate transporter in ArabidopsisENTakaakiMiyajiTakashiKuromoriYuTakeuchiNaokiYamajiKengoYokoshoAtsushiShimazawaErikoSugimotoHiroshiOmoteJian FengMaKazuoShinozakiYoshinoriMoriyamaAscorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama2045-232242014Identification of a mammalian vesicular polyamine transporterENMikiHiasaTakaakiMiyajiYukaHarunaTomoyaTakeuchiYuikaHaradaSawakoMoriyamaAkitsuguYamamotoHiroshiOmoteYoshinoriMoriyamaSpermine 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).No potential conflict of interest relevant to this article was reported.Acta Medica Okayama105152008Identification of a vesicular nucleotide transporter56835686ENKeisukeSawadaNorikoEchigoNarinobuJugeTakaakiMiyajiMasatoOtsukaHiroshiOmoteAkitsuguYamamotoYoshinoriMoriyama<p>ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission. </p>No potential conflict of interest relevant to this article was reported.European Molecular Biology OrganizationActa Medica Okayama0261-418925182006Secretion of L-glutamate from osteoclasts through transcytosis41754186ENRiyoMorimotoShunsukeUeharaShoukiYatsushiroNarinobuJugeZhaolinHuaShigenoriSenohNorikoEchigoMitsukoHayashiToshihideMizoguchiTadashiNinomiyaNobuyukiUdagawaHiroshiOmoteAkitsuguYamamotoRobert HEdwardsYoshinoriMoriyamaOsteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama102502007A human transporter protein that mediates the final excretion step for toxic organic cations1792317928ENMasatoOtsukaTakuyaMatsumotoRiyoMorimotoShigeoAriokaHiroshiOmoteYoshinoriMoriyama<p>In mammals, toxic electrolytes of endogenous and exogenous origin are excreted through the urine and bile. Before excretion, these compounds cross numerous cellular membranes in a transporter-mediated manner. However, the protein transporters involved in the final excretion step are poorly understood. Here, we show that MATE1, a human and mouse orthologue of the multidrug and toxin extrusion (MATE) family conferring multidrug resistance on bacteria, is primarily expressed in the kidney and liver, where it is localized to the luminal membranes of the urinary tubules and bile canaliculi. When expressed in HEK293 cells, MATE1 mediates H+-coupled electroneutral exchange of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP). Its substrate specificity is similar to those of renal and hepatic H+-coupled organic cations (OCs) export. Thus, MATE1 appears to be the long searched for polyspecific OC exporter that directly transports toxic OCs into urine and bile.</p>No potential conflict of interest relevant to this article was reported.The Company of Biologists LimitedActa Medica Okayama0022-094920312000Luminal acidification of diverse organelles by V-ATPase in animal cells107116ENMasamitsuFutaiToshihikoOkaGe-hongSun-WadaYoshinoriMoriyamaHiroshiKanazawaYohWadaEukaryotic cells contain organelles bounded by a single membrane in the cytoplasm. These organelles have differentiated to carry out various functions in the pathways of endocytosis and exocytosis. Their lumina are acidic, with pH ranging from 4.5 to 6.5. This article describes recent studies on these animal cell organelles
focusing on (1) the primary proton pump (vacuolar-type H+-ATPase) and (2) the functions of the organelle luminal acidity. We also discuss similarities and differences between vacuolar-type H+-ATPase and F-type ATPase. Our own studies and interests are emphasized.No potential conflict of interest relevant to this article was reported.The Company of Biologists LimitedActa Medica Okayama0022-094920312001Synaptic-like microvesicles, synaptic vesicle counterparts in endocrine cells, are involved in a novel regulatory mechanism for the synthesis and secretion of hormones117125ENYoshinoriMoriyamaMitsukoHayashiHiroshiYamadaShoukiYatsushiroShougoIshioAkitsuguYamamotoMicrovesicles in endocrine cells are the morphological and functional equivalent of neuronal synaptic vesicles. Microvesicles accumulate various neurotransmitters through a transmitter-specific vesicular transporter energized by vacuolar H+-ATPase. We found that mammalian pinealocytes, endocrine cells that synthesize and secrete melatonin, accumulate L-glutamate in their microvesicles and secrete it through exocytosis. Pinealocytes use L-glutamate as either a paracrine- or autocrine-like chemical transmitter in a receptor-mediated manner, resulting in inhibition of melatonin synthesis. In this article, we briefly describe the overall features of the microvesicle-mediated signal-transduction mechanism in the pineal gland and discuss the important role of acidic organelles in a novel regulatory mechanism for hormonal synthesis and secretion.No potential conflict of interest relevant to this article was reported.