Activation of Ionic Channels during Regulatory Volume Decrease

在低渗浴溶液中，培养的肠上皮细胞(肠407)表现出最初的渗透性肿胀和随后的调节性体积减少(RVD)。细胞内记录显示，在对照离子条件下(4.2 mm&lt；K^+&gt；，60 mm&lt；Cl^-&gt；)，RVD与双相超极化有关。应用&lt；Ca^(2+)&gt；激活的&lt；K^+&gt；通道的抑制剂奎宁可阻断超极化。在低-lt；Cl^-&gt；溶液(4.2 mM；K^+&gt；，6 mM；Cl^-&gt；)中，渗透压激发引起一种特征的双相反应；即，短暂的奎宁敏感的超极化之后是延长的去极化。去极化可被氯离子通道阻断剂SITS阻断，但不能被奎宁阻断。双微电极电压钳研究表明，由a-lt；K^+&gt；电流和a-lt；Cl^-gt；电流组成的双相膜电流与RVD过程中膜电位的变化有关。为了确定细胞内&lt；Ca^(2+)&gt；离子激活的电流，进行了细胞内&lt；Ca^(2+)&gt；离子注入和&lt；Ca^(2+)&gt；选择性微电极研究。向细胞内微量注射~lt；Ca^(2+)&gt；离子引起短暂的单相超极化，这种超极化对奎宁(而不是SITS)敏感，但在低&lt；Cl2-&gt；溶液中不会引起随后的去极化。在低&lt；Cl-&gt；条件下RVD过程中的细胞内&lt；Ca^(2+)&gt；测量表明，胞浆游离&lt；Ca^(2+)&gt；浓度的显著增加与初始超极化(&lt；K^+&gt；电流激活)有关，而与随后的去极化(&lt；Cl^-&>t；电流激活)无关。根据这些观察结果，我们得出结论：在肠道407细胞的RVD过程中，存在着独立的导电通道&lt；K^+&gt；和&lt；Cl-&gt；通道，前者被胞浆内游离的&lt；Ca^(2+)&gt；离子激活，而后者则不是。

During exposure to a hypotonic bathing solution, cultured epithelial cells (Intestine 407) exhibited initial osmotic swelling and a subsequent regulatory volume decrease (RVD). Intracellular recordings showed that the RVD was associated with biphasic hyperpolarizations under a control ionic condition (4.2 mM <K^+> , 60 mM <Cl^-> ). The hyperpolarizations were blocked by the application of quinine, an inhibitor of <Ca^(2+)> - activated <K^+> channels. In a low <Cl^-> solution (4.2 mM <K^+> , 6 mM <Cl^-> ), the osmotic challenge provoked a characteristic biphasic response; that is, a transient, quinine-sensitive hyperpolarization was followed by a prolonged depolarization. The depolarization was blocked by SITS, a blocker of <Cl^-> channels, but not by quinine. Two-microelectrode voltage clamp studies indicated that biphasic membrane currents consisting of a <K^+> current and a <Cl^-> current were associated with the membrane potential changes upon the RVD process. To determine which current is activated by intracellular <Ca^(2+)> ions, studies by intracellular <Ca^(2+)> injections and with <Ca^(2+)> -selective microelectrodes were carried out. Electrophoretic microinjection of <Ca^(2+)> ions into the cell evoked a transient monophasic hyperpolarization, which was sensitive to quinine (not SITS), but never induced a subsequent depolarization in a low <Cl^-> solution. Intracellular <Ca^(2+)> measurements during the RVD process under a low <Cl^-> condition demonstrated that a sizable increase in the cytosolic free <Ca^(2+)> concentration was associated with an initial hyperpolarization ( <K^+> current activation) but not with a subsequent depolarization ( <Cl^-> current activation). Based on these observations, it is concluded that separate electroconductive pathways, <K^+> and <Cl^-> channels, are operating during the RVD process in Intestine 407 cells, and that the former is activated by increased cytoplasmic free <Ca^(2+)> ions but the latter is not.