| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1986: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1985: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
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.
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