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Department of Physiology, Faculty of Medicine, Kyoto University, Japan.
1. During exposure to a hypotonic solution, cultured human epithelial cells (Intestine 407) exhibited a regulatory volume decrease (RVD) after initial osmotic swelling. 2. The volume readjustment was slowed by elevating the extracellular K+ concentration and facilitated by reducing the extracellular Cl- concentration. Not only putative K+ channel blockers, quinine and Ba2+, but also a stilbene derivative Cl- channel blocker (SITS) inhibited the RVD. 3. The volume recovery of hypoosmotically swollen cells was very much suppressed by the deprivation of extracellular Ca2+ ions or by chelation of cytosolic Ca2+ ions with Quin-2 loaded within the cells. 4. Biphasic membrane potential changes were associated with the RVD process at low extracellular K+ and Cl- concentrations. The initial hyperpolarizing response was inhibited by quinine and Ba2+, whereas the late depolarizing response was inhibited by SITS. The deprivation of extracellular Ca2+ inhibited the initial hyperpolarizing phase but not the late depolarizing phase. 5. Two-microelectrode voltage clamp studies showed that the initial hyperpolarization and late depolarization were associated with quinine-sensitive outward currents and SITS-sensitive inward currents, respectively. The reversal potentials estimated from the current-voltage curves were about -80 mV for the initial response and -27 mV for the late response. Tenfold changes in the K+ and Cl- concentrations shifted these reversal potentials by 50 mV for the initial response and by 42 mV for the late response. 6. Under whole-cell recordings, similar current changes were observed in the cells exposed to a hypotonic solution, when the intracellular Ca2+ ions were moderately buffered with 1 mM-EGTA in the dialysing solution filled in a patch pipette. When most Ca2+ ions were chelated with 10 mM-EGTA in the pipette solution, the initial outward current as well as the corresponding hyperpolarization was suppressed, but the late current associated with the depolarizing phase was preserved. 7. Intracellular Ca2+ injections induced an increase in the quinine-sensitive K+ conductance but failed to activate the Cl- conductance. 8. It is concluded that both K+ and Cl- channels are involved in the regulatory volume decrease, and that the former channel is exclusively activated by elevation of the cytosolic Ca2+ concentration in the epithelial cells.
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