Hypotonicity activates volume-sensitive Cl- currents, which are implicated in the regulatory volume decrease (RVD) responses and transport of taurine in human cervical cancer HT-3 cells. In this study, the role of cytoskeleton in the regulation of volume-sensitive Cl- channels and RVD responses in HT-3 cells was studied. Cells were incubated with various compounds, which depolymerized or polymerized cytoskeletal elements, i.e. actin filaments and microtubules. The hypotonicity-induced changes in Cl- conductance and in cell volume were measured by whole-cell voltage clamping and cell size monitoring, respectively. Our results show that in HT-3 cells hypotonicity activated an outward rectified Cl- current that was abrogated by Cl- channel blockers. Cytochalasin B, an actin-depolymerizing compound, induced a substantial increase in Cl- conductance under isotonic condition and potentiated the expression of Cl- currents in hypotonic stress. Phorbol 12-myristate 13-acetate (PMA) significantly inhibited the cytochalasin B-induced activation of Cl- conductance under isotonic condition. On the other hand, treatment with cytochalasin B significantly prolonged the RVD responses. Phalloidin, a stabilizer of actin polymerization, did not change the basal currents under isotonic condition, but completely abolished the increase in whole-cell Cl- conductance elicited by hypotonicity and retarded the cell volume recovery. Colchicine, a microtubule-assembly inhibitor, had no effect on either basal Cl- conductance or volume-sensitive Cl- current and was unable to inhibit the RVD responses. Taxol, a microtubule-stabilizing compound, did not alter the basal Cl- conductance, but inhibited the activation of volume-sensitive Cl- channels as well as the process of RVD in a dose-dependent manner. These data support the notion that functional integrity of actin filaments and microtubules plays critical roles in maintaining the RVD responses and activation of Cl- channels in human cervical cancer HT-3 cells.
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