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Involvement of PKC-alpha in regulatory volume decrease responses and activation of volume-sensitive chloride channels
1Department of Obstetrics and Gynecology, National Cheng Kung University Medical College, Tainan 704, Taiwan. chougyn@mail.ncku.edu.tw
Abstract:
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.
Insights
Protein kinase C-alpha (PKC-alpha) and extracellular calcium are key to regulatory volume decrease (RVD) and volume-sensitive chloride channels in HT-3 cervical cancer cells.
Area of Science:
- Cellular Biology
- Molecular Biology
- Oncology
Background:
- Regulatory volume decrease (RVD) is a critical cellular process for maintaining cell volume homeostasis.
- Volume-sensitive chloride channels play a significant role in RVD.
- Protein kinase C (PKC) isoforms are implicated in various cellular signaling pathways, but their specific roles in RVD and chloride channel activation require elucidation.
Purpose of the Study:
- To identify the specific PKC isoform involved in RVD in HT-3 cervical cancer cells.
- To investigate the signal transduction pathways regulating volume-sensitive chloride channels.
- To determine the role of calcium (Ca2+) in RVD and chloride current activation.
Main Methods:
- Microinjection of PKC isoform-specific antibodies and EDTA.
- Immunofluorescence staining to assess protein translocation.
- Western blot analysis for protein expression and phosphorylation.
- Whole-cell patch-clamp electrophysiology.
- Pharmacological inhibition and activation of PKC.
Main Results:
- PKC-alpha antibody prolonged RVD, unlike PKC-beta or PKC-gamma antibodies.
- Hypotonic stress induced PKC-alpha translocation to the cell membrane, with increased membrane-associated PKC-alpha levels and phosphorylation.
- PKC blockers and PKC-alpha antibody inhibited volume-sensitive chloride currents, while PMA reversed neomycin's inhibition.
- Chloride current activation was independent of intracellular Ca2+ but required extracellular Ca2+.
Conclusions:
- PKC-alpha is critically involved in mediating RVD responses in HT-3 cells.
- Extracellular Ca2+ is essential for the activation of volume-sensitive chloride channels.
- PKC-alpha activation and extracellular Ca2+ are key components of the signaling pathway for RVD and chloride channel function in this cancer cell line.