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Involvement of PKC- in regulatory volume decrease responses and activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells

Cheng-Yang Chou, Meng-Ru Shen, Kuei-Sen Hsu *, Hsueh-Yin Huang and Hui-Chuan Lin

1. The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells. The role of Ca²⁺ in RVD and in the activation of chloride currents was also studied.

2. The time course of RVDs was prolonged by microinjection of PKC- antibody but not by PKC- or PKC- antibody, and also by exposure to Ca²⁺-free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC- to the cell membrane, whereas PKC- or PKC- remained unaffected. The translocation of PKC- was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC- level in terms of intensity and phosphorylation in the cell membrane, while neither PKC- nor PKC- was activated upon hyposmotic challenge.

3. Whole-cell patch-clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume-sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12-myristate, 13-acetate (PMA). Moreover, the PKC inhibitor and PKC- antibody, but not PKC- or PKC- antibody, significantly attenuated the chloride currents. The activation of volume-sensitive chloride currents were insensitive to the changes of intracellular Ca²⁺ but required the presence of extracellular Ca²⁺.

4. Our results suggest the involvement of PKC- and extracellular Ca²⁺ in RVD responses and the activation of volume-sensitive chloride channels in HT-3 cells.

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