論文 - 緒方　元気

Theoretical and experimental analysis of ototoxic mechanism in the spiral ligament fibrocytes by multi-level simulation with ion transports in the cochlea.

Nin F, Yoshida T, Murakami S, Uetsuka S, Ogata G, Kurachi Y, Hibino H

Nihon yakurigaku zasshi. Folia pharmacologica Japonica 147 （ 2 ） 80 - 3 2016年02月

ISSN  0015-5691

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Molecular architecture of the stria vascularis membrane transport system, which is essential for physiological functions of the mammalian cochlea.

Uetsuka S, Ogata G, Nagamori S, Isozumi N, Nin F, Yoshida T, Komune S, Kitahara T, Kikkawa Y, Inohara H, Kanai Y, Hibino H

The European journal of neuroscience （WILEY-BLACKWELL）  42 （ 3 ） 1984 - 2002 2015年08月

査読有り,  ISSN  0953-816X

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Stria vascularis of the mammalian cochlea transports K+ to establish the electrochemical property in the endolymph crucial for hearing. This epithelial tissue also transports various small molecules. To clarify the profile of proteins participating in the transport system in the stria vascularis, membrane components purified from the stria of adult rats were analysed by liquid chromatography tandem mass spectrometry. Of the 3236 proteins detected in the analysis, 1807 were membrane proteins. Ingenuity Knowledge Base and literature data identified 513 proteins as being expressed on the plasma membrane', these included 25 ion channels and 79 transporters. Sixteen of the former and 62 of the latter had not yet been identified in the stria. Unexpectedly, many Cl- and Ca2+ transport systems were found, suggesting that the dynamics of these ions play multiple roles. Several transporters for organic substances were also detected. Network analysis demonstrated that a few kinases, including protein kinase A, and Ca2+ were key regulators for the strial transports. In the library of channels and transporters, 19 new candidates for uncloned deafness-related genes were identified. These resources provide a platform for understanding the molecular mechanisms underlying the epithelial transport essential for cochlear function and the pathophysiological processes involved in hearing disorders.

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NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K⁺ transport that controls the electrochemical properties in the mammalian cochlea.

Yoshida T, Nin F, Ogata G, Uetsuka S, Kitahara T, Inohara H, Akazawa K, Komune S, Kurachi Y, Hibino H

Pflugers Archiv : European journal of physiology （SPRINGER）  467 （ 7 ） 1577 - 1589 2015年07月

査読有り,  ISSN  0031-6768

　概要を見る

Unidirectional K+ transport across the lateral cochlear wall contributes to the endocochlear potential (EP) of +80 mV in the endolymph, a property essential for hearing. The wall comprises two epithelial layers, the syncytium and the marginal cells. The basolateral surface of the former and the apical membranes of the latter face the perilymph and the endolymph, respectively. Intrastrial space (IS), an extracellular compartment between the two layers, exhibits low [K+] and a potential similar to the EP. This IS potential (ISP) dominates the EP and represents a K+ diffusion potential elicited by a large K+ gradient across the syncytial apical surface. The K+ gradient depends on the unidirectional K+ transport driven by Na+,K+-ATPases on the basolateral surface of each layer and the concomitant Na+,K+,2Cl(-)-cotransporters (NKCCs) in the marginal cell layer. The NKCCs coexpressed with the Na+,K+-ATPases in the syncytial layer also seem to participate in the K+ transport. To test this hypothesis, we examined the electrochemical properties of the lateral wall with electrodes measuring [K+] and potential. Blocking NKCCs by perilymphatic perfusion of bumetanide suppressed the ISP. Unexpectedly and unlike the inhibition of the syncytial Na+,K+-ATPases, the perfusion barely altered the electrochemical properties of the syncytium but markedly augmented [K+] of the IS. Consequently, the K+ gradient decreased and the ISP declined. These observations resembled those when the marginal cells' Na+,K+-ATPases or NKCCs were blocked with vascularly applied inhibitors. It is plausible that NKCCs in the marginal cells are affected by the perilymphatically perfused bumetanide, and these transporters, but not those in the syncytium, mediate the unidirectional K+ transport.

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内耳の特殊体液の恒常性が立脚する上皮イオン輸送の分子構築の解析

上塚 学, 緒方 元気, 永森 收志, 五十棲 規嘉, 吉田 崇正, 任 書晃, 北原 糺, 吉川 欣亮, 猪原 秀典, 金井 好克, 日比野 浩

日本プロテオーム学会大会要旨集 （日本プロテオーム学会（日本ヒトプロテオーム機構））  2015   191 - 191 2015年

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Functional analysis of Na+,K+,2Cl(-)-cotransporters contributing to the endocochlear potential

Yoshida, Takamasa and Nin, Fumiaki and Ogata, Genki and Uetsuka, Satoru and Komune, Shizuo and Kurachi, Yoshihisa and Hibino, Hiroshi

Journal of Pharmacological Sciences 124   138P - 138P 2014年

ISSN  1347-8613

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.

Adachi N, Yoshida T, Nin F, Ogata G, Yamaguchi S, Suzuki T, Komune S, Hisa Y, Hibino H, Kurachi Y

The Journal of physiology （WILEY-BLACKWELL）  591 （ 18 ） 4459 - 72 2013年09月

査読有り,  ISSN  0022-3751

　概要を見る

The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

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Dopamine and full-field illumination activate D1 and D2-D5-type receptors in adult rat retinal ganglion cells

Ogata, G. and Stradleigh, T.W. and Partida, G.J. and Ishida, A.T.

Journal of Comparative Neurology （WILEY-BLACKWELL）  520 （ 17 ） 4032 - 4049 2012年12月

査読有り,  ISSN  0021-9967

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Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor-driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor-driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch-clamp recordings, Ca2+ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light-induced rises in cAMP, light-induced activation of Ca2+/calmodulin-dependent protein kinase II, and dopamine-induced Ca2+ influx; and 4) the Ca2+ rise is markedly reduced by removing extracellular Ca2+ and by an IP3 receptor antagonist (2-APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP-dependent protein kinase to modulate retinal ganglion cell excitability. J. Comp. Neurol. 520: 4032-4049, 2012. (C) 2012 Wiley Periodicals, Inc.

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Thy1 associates with the cation channel subunit HCN4 in adult rat retina.

Partida GJ, Stradleigh TW, Ogata G, Godzdanker I, Ishida AT

Investigative ophthalmology & visual science （ASSOC RESEARCH VISION OPHTHALMOLOGY INC）  53 （ 3 ） 1696 - 703 2012年03月

査読有り,  ISSN  0146-0404

　概要を見る

PURPOSE. The membrane expression and gene promoter of the glycosylphosphatidylinositol (GPI)-anchored protein Thy1 have been widely used to examine the morphology and distribution of retinal ganglion cells in normal eyes and disease models. However, it is not known how adult mammalian retinal neurons use Thy1. Because Thy1 is not a membrane-spanning protein and, instead, complexes with structural and signaling proteins in other tissues, the aim of this study was to find protein partners of retinal Thy1.
METHODS. Coimmunoprecipitation, immunohistochemistry, confocal imaging, and patch-clamp recording were used to test for association of Thy1 and HCN4, a cation channel subunit, in adult rat retina.
RESULTS. Hyperpolarization of cells immunopanned by an anti-Thy1 antibody activated HCN channels. Confocal imaging showed that individual somata in the ganglion cell layer bound antibodies against Thy1 and HCN4, that the majority of these bindings colocalized, and that some of the immunopositive cells also bound antibody against a ganglion cell marker (Brn3a). Consistent with these results, Thy1 and HCN4 were coimmunoprecipitated by magnetic beads coated with either anti-Thy1 antibody or anti-HCN4 antibody. In control experiments, beads coated with these antibodies did not immunoprecipitate a photoreceptor rim protein (ABCR) and uncoated beads did not immunoprecipitate either Thy1 or HCN4.
CONCLUSIONS. This is the first report that Thy1 colocalizes and coimmunoprecipitates with a membrane-spanning protein in retina, that Thy1 complexes with an ion channel protein in any tissue, and that a GPI-anchored protein associates with an HCN channel subunit protein. (Invest Ophthalmol Vis Sci. 2012; 53: 1696-1703) DOI:10.1167/iovs.11-9307

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Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells.

Stradleigh TW, Ogata G, Partida GJ, Oi H, Greenberg KP, Krempely KS, Ishida AT

The Journal of comparative neurology （WILEY-BLACKWELL）  519 （ 13 ） 2546 - 73 2011年09月

査読有り,  ISSN  0021-9967

　概要を見る

The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I-h) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I-h in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I-h less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I-h activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I-h properties with the previously reported presence of HCN4 in these cells, and indicate that I-h is biophysically similar in morphologically diverse retinal ganglion cells and differs from I-h in rods, cones, and bipolar cells. J. Comp. Neurol. 519:2546-2573, 2011. (C) 2011 Wiley-Liss, Inc.

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Inhibition of adult rat retinal ganglion cells by D1-type dopamine receptor activation.

Hayashida Y, Rodríguez CV, Ogata G, Partida GJ, Oi H, Stradleigh TW, Lee SC, Colado AF, Ishida AT

The Journal of neuroscience : the official journal of the Society for Neuroscience （SOC NEUROSCIENCE）  29 （ 47 ） 15001 - 16 2009年11月

査読有り,  ISSN  0270-6474

　概要を見る

The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D-1a receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D-1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D-1-type receptors, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking I-h. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (I-Na) amplitude, and tetrodotoxin, at doses that reduced I-Na as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D-1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the presynaptic and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing.

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The frequency of interictal activity was modulated by the generation of the activity per se

Ogata Genki, Natsume Kiyohisa

NEUROSCIENCE RESEARCH 58   S238 - S238 2007年

ISSN  0168-0102

Synchronization Process of Neurons Depends on Giant EPSP of Hippocampal Pyramidal Cells

OGATA Genki, NATSUME Kiyohisa

J Signal Process （〔信号処理学会〕）  10 （ 6 ） 399 - 406 2006年11月

ISSN  1342-6230

Dependence of Neuronal Synchronization Process on the Giant EPSP Pattern of Hippocampal Pyramidal Cells

OGATA Genki, NATSUME Kiyohisa

J Signal Process 10 （ 4 ） 239 - 242 2006年07月

ISSN  1342-6230