{"_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": ["22"]}, "author_link": ["9343", "9344", "9342"], "item_1_biblio_info_21": {"attribute_name": "書誌情報（ソート用）", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2000-03-24", "bibliographicIssueDateType": "Issued"}, "bibliographic_titles": [{}]}]}, "item_1_creator_2": {"attribute_name": "著者名", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "清水, 貴浩"}], "nameIdentifiers": [{"nameIdentifier": "9342", "nameIdentifierScheme": "WEKO"}]}]}, "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": [{"nameIdentifier": "9343", "nameIdentifierScheme": "WEKO"}]}]}, "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_1": {"attribute_name": "ID", "attribute_value_mlt": [{"subitem_description": "2000041", "subitem_description_type": "Other"}]}, "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). \u003cbr /\u003eRT-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.\u003cbr /\u003eI 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\u0027,N\u0027-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.\u003cbr /\u003eEffects 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. \u003cbr /\u003eExtracellular Ca2+ or Mg2+ exhibited an additional effect on the volume-sensitive Cl- current: facilitation of its depolarization-induced \u003cbr /\u003einactivation 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. \u003cbr /\u003eTaken 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_1_text_20": {"attribute_name": "業務メモ", "attribute_value_mlt": [{"subitem_text_value": "（2018年2月15日）本籍など個人情報の記載がある旧要旨・審査要旨を個人情報のない新しいものに差し替えた。承諾書等未確認。要確認該当項目修正のこと。"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "SHIMIZU, Takahiro", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "9344", "nameIdentifierScheme": "WEKO"}]}]}, "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", "download_preview_message": "", "file_order": 0, "filename": "甲477_要旨.pdf", "filesize": [{"value": "307.5 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 307500.0, "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", "download_preview_message": "", "file_order": 1, "filename": "甲477_本文.pdf", "filesize": [{"value": "1.5 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 1500000.0, "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"], "permalink_uri": "https://ir.soken.ac.jp/records/1108", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "1108", "relation": {}, "relation_version_is_last": true, "title": ["Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells"], "weko_shared_id": 1}