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PubMed · 9763633

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

C Y Chou·M R Shen·K S Hsu·H Y Huang·H C Lin
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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.

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Blotting, WesternCalcium SignalingCell SizeChloride ChannelsElectric StimulationElectrophysiologyFemaleFluorescent Antibody Technique, DirectHumansIsoenzymesMembrane PotentialsPatch-Clamp TechniquesProtein Kinase CProtein Kinase C-alphaSignal TransductionSubcellular FractionsTumor Cells, CulturedUterine Cervical NeoplasmsJournal ArticleResearch Support, Non-U.S. Gov't

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C Y Chou, M R Shen, K S Hsu, H Y Huang, H C Lin. 1998-10-15. Involvement of PKC-alpha in regulatory volume decrease responses and activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells.. https://doi.org/10.1111/j.1469-7793.1998.435be.x

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