{"created":"2023-06-20T13:21:01.287373+00:00","id":1108,"links":{},"metadata":{"_buckets":{"deposit":"3d329cfe-0ab3-4549-adf1-62a2a8f98bb1"},"_deposit":{"created_by":1,"id":"1108","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1108"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001108","sets":["2:430:22"]},"author_link":["9343","9344","9342"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"清水, 貴浩"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"シミズ, タカヒロ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2000-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"Since extracellular Ca2+ has been reported to modulate swelling-activated Cl- currents, I examined an involvement of G protein-coupled Ca2+-sensing receptor (CaR) in the regulation of the volume-sensitive Cl- channel by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting and whole-cell patch-clamp techniques, in a human epithelial cell line (Intestine 407). <br />RT-PCR confirmed that the Intestine 407 cell contains mRNAs cording for the CaR, and expression of the CaR protein was evidenced by immunoblotting analysis. The swelling-activated whole-cell Cl- current was augmented by addition of Ca2+ to the bathing solution in a concentration-dependent manner. The total Ca2+ concentration for half-maximal stimulation (EC50) was 6.5 mM. A rise in the extracellular Mg2+ concentration also concentration-dependently increased the amplitude of volume-sensitive Cl- currents, though less effective (EC50 of around 22 mM) than Ca2+. In addition, other CaR agonists, La3+ (3 μM), neomycin (500 μM) and spermine (1 mM), significantly augmented the Cl- current. To further confirm an involvement of the CaR in the upregulating effect of extracellular Ca2+ on the volume-sensitive Cl- current, I examined the effects of GDPβS, which is a G protein inhibitor, and GTPγS, which is a G protein activator, on the Cl- current. Incorporation of GDPβS in the pipette (intracellular) solution abolished extracellular Ca2+-induced enhancement of the Cl- current. Under Ca2＋ - and Mg2+ -free conditions, the amplitude of volume-sensitive Cl- currents became increased by the presence of intracellular GTPγS. Further augmentation was never induced by addition of extracellular Ca2+ in the presence of intracellular GTPγS. These results demonstrate that the G protein-coupled CaR mediates Ca2+-induced upregulation of the volume-sensitive Cl- channel in Intestine 407 cells.<br />I then investigated the signal transduction pathway of CaR-mediated regulation of volume-sensitive Cl- channel. The augmenting effect of extracellular Ca2+ on the Cl- current could be abolished neither by application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA: 5 mM) to the pipette (intracellular) solution nor by 24 h-pretreatment with purtussis toxin (PTX: 100 ng/ml). When the intracellular cAMP concentration was elevated by application of a cocktail of forskolin (10 μM), dibutylyl cAMP (1 mM) and 3-isobutyl-1-methylxanthine (IBMX: 400 μM), the amplitude of volume-sensitive Cl- current was markedly enlarged. Under the cAMP stimulation, extracellular Ca2+ failed to increase the Cl- current. These results suggest that the CaR is coupled to Gs and modulates the volume-sensitive Cl- channel via an increase in intracellular cAMP level.<br />Effects of CaR stimulation on volume sensitivity of swelling-activated whole-cell Cl- current was then assessed. When the whole-cell Cl- current density was plotted against the relative cell surface area measured simultaneously, the data points were well fitted to the Boltzmann function. Elevation of extracellular Ca2+ shifted the curve to the left and increased the slope. These results indicate that CaR-mediated augmentation of the Cl- channel is due to increased sensitivity of the channel (or its accessory volume sensor) to cell volume expansion. <br />Extracellular Ca2+ or Mg2+ exhibited an additional effect on the volume-sensitive Cl- current: facilitation of its depolarization-induced <br />inactivation kinetics. The inactivation time course of the Cl- current at large positive potentials became faster in the presence of extracellular Ca2+ or Mg2+. The relative half inactivation time at + 100 mV was maximally decreased to 60.4 and 40.7% by Ca2+ and Mg2+, respectively. In contrast to the effects on the Cl- current amplitude, EC50 of the Mg2+ effect on the inactivation kinetics (2.1 mM) was smaller than that of the Ca2+ effect (2.5 mM). In addition, all other CaR agonists examined failed to accelerate the inactivation time course. Furthermore, the extracellular Ca2+ effect on inactivation kinetics was not affectcd by GDPβS or GTPγS. These results indicate that the CaR does not mediate the effect of extracellular Ca2+ or Mg2+ on the depolarization-induced inactivation kinetics. <br />Taken together, it is concluded that stimulation of CaR induces upregulation of volume-sensitive Cl- channels by enhancing the volume expansion sensitivity in human epithelial Intestine 407 cells. The second messenger is likely to be cAMP but not Ca2+ in the cytosol. The accelerating effect of extracellular divalent cations on inactivation time course of the Cl- current is induced by a different mechanism without mediation by the CaR.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第477号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"20 生理科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"1999"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"SHIMIZU, Takahiro","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲477_要旨.pdf","filesize":[{"value":"307.5 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/1108/files/甲477_要旨.pdf"},"version_id":"b5fe0f57-0034-478a-a9dc-f90ff8a46f89"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲477_本文.pdf","filesize":[{"value":"1.5 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/1108/files/甲477_本文.pdf"},"version_id":"54049ae9-ca72-4204-b0f0-7119ffaf4f13"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells"},{"subitem_title":"Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["22"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1108","relation_version_is_last":true,"title":["Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:47:48.524240+00:00"}