WEKO3
アイテム
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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. 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Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells
https://ir.soken.ac.jp/records/1108
https://ir.soken.ac.jp/records/1108074a8b4e-1aaa-4f95-8ec5-ff8aeae66dce
名前 / ファイル | ライセンス | アクション |
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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公開日 | 2010-02-22 | |||||
タイトル | ||||||
タイトル | Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells | |||||
タイトル | ||||||
言語 | en | |||||
タイトル | Ca2+-sensing receptor-mediated regulation ofvolume-sensitive Cl-channels in human epithelial cells | |||||
言語 | ||||||
言語 | eng | |||||
資源タイプ | ||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
資源タイプ | thesis | |||||
著者名 |
清水, 貴浩
× 清水, 貴浩 |
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フリガナ |
シミズ, タカヒロ
× シミズ, タカヒロ |
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著者 |
SHIMIZU, Takahiro
× SHIMIZU, Takahiro |
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学位授与機関 | ||||||
学位授与機関名 | 総合研究大学院大学 | |||||
学位名 | ||||||
学位名 | 博士(理学) | |||||
学位記番号 | ||||||
内容記述タイプ | Other | |||||
内容記述 | 総研大甲第477号 | |||||
研究科 | ||||||
値 | 生命科学研究科 | |||||
専攻 | ||||||
値 | 20 生理科学専攻 | |||||
学位授与年月日 | ||||||
学位授与年月日 | 2000-03-24 | |||||
学位授与年度 | ||||||
1999 | ||||||
要旨 | ||||||
内容記述タイプ | Other | |||||
内容記述 | 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. | |||||
所蔵 | ||||||
値 | 有 | |||||
フォーマット | ||||||
内容記述タイプ | Other | |||||
内容記述 | application/pdf |