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) 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-alpha antibody but not by PKC-beta or PKC-gamma 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-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 returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC-alpha level in terms of intensity and phosphorylation in the cell membrane, while neither PKC-beta nor PKC-gamma 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-alpha antibody, but not PKC-beta or PKC-gamma 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-alpha and extracellular Ca2+ in RVD responses and the activation of volume-sensitive chloride channels in HT-3 cells.
Chou
C Y
CY
Department of Obstetrics and Gynecology, National Cheng Kung University Medical College, Tainan 704, Taiwan. chougyn@mail.ncku.edu.tw
Shen
M R
MR
Hsu
K S
KS
Huang
H Y
HY
Lin
H C
HC
D015153:Blotting, Western; D020013:Calcium Signaling / Q000502:physiology; D048429:Cell Size; D018118:Chloride Channels / Q000502:physiology*; D004558:Electric Stimulation; D004594:Electrophysiology; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000502:physiology*; D051571:Protein Kinase C-alpha; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions / Q000502:physiology; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000473:pathology
D018118:Chloride Channels; D007527:Isoenzymes; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha