INVOLVEMENT OF PKC-ALPHA IN REGULATORY VOLUME DECREASE RESPONSES AND ACTIVATION OF VOLUME-SENSITIVE CHLORIDE CHANNELS IN HUMAN CERVICAL-CANCER HT-3 CELLS

1. The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) re sponses, and to investigate the signal transduction pathways underlyin g the activation of volume-sensitive chloride channels in human cervic al 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 pr olonged by microinjection of PKC-alpha antibody but not by PKC-beta or PKC-gamma antibody, and also by exposure to Ca2+-free medium, in part icular when combined with microinjection of EDTA. Immunofluorescence s taining showed that hypotonic superfusion evoked the translocation of PRC-alpha to the cell membrane, whereas PKC-beta or PKC-gamma remained unaffected. The translocation of PKC-alpha was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and return ed to the cytoplasm 60 min after hypotonic exposure. Western blot anal yses showed an increased PKC-alpha level in terms of intensity and pho sphorylation in the cell membrane, while neither PKC-beta nor PKC-gamm a was activated upon hyposmotic challenge. 3. Whole-cell patch-clamp s tudies demonstrated that neomycin and PKC: blockers such as staurospor ine and H7 inhibited volume-sensitive chloride currents. The inhibitor y effect of neomycin on chloride currents can be reversed by the PKC: activator phorbol 12-myristate, 13-acetate (PMA). Moreover, the PKC in hibitor and PKC-alpha antibody, but not PKC-beta or PKC-gamma antibody , significantly attenuated the chloride currents. The activation of vo lume-sensitive chloride currents were insensitive to the changes of in tracellular Ca2+ but required the presence of extracellular Ca2+. 4. O ur results suggest the involvement of PRC-alpha and extracellular Ca2 in RVD responses and the activation of volume-sensitive chloride chan nels in HT-3 cells.