Effect of PMA on the integrity of the membrane skeleton and morphology of epithelial MDCK cells is dependent on the activity of amiloride-sensitive ion transporters and membrane potential.

: The signaling pathways from an activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) to the rearrangement of actin-based cytoskeleton and membrane skeleton of epithelial MDCK cells were studied by visualizing the cytoskeletal organization with immunofluorescence microscopy and by measuring intracellular pH, sodium ion concentration and membrane potential with the aid of fluorescent intracellular indicators. Upon PMA treatment the MDCK cells lost their cubic shape and acquired a spindle-like morphology. The stress fibers were depolymerized, and fodrin, the main component of the membrane skeleton, was released from the lateral walls to the cytosol. These changes were accompanied by depolarization of the cells, decrease in the intracellular pH and sodium ion concentration. In order to test the mutual correlation between the PMA-induced alterations we treated the cells with PMA in the presence of channel inhibitors or ionophores and in defined media. The effects of PMA on the membrane skeleton and morphology could be reversed in media lacking Na+ or K+ ions or by hyperpolarizing agents, dimethylamiloride and valinomycin, suggesting that the effects of PMA on the cytoskeleton were dependent on the ion gradients and membrane potential across the cell membrane. Moreover, the morphological changes and instabilization of the membrane skeleton of MDCK cells took place spontaneously without PMA in depolarizing conditions, in potassium gluconate buffer. We suggest that the membrane potential across the cell membrane of MDCK cells together with the activity of amiloride-sensitive cation transporters transmits signals in the protein kinase C (PKC) pathway leading from activation of PKC to fibroblast-like morphology and cytoplasmic localization of membrane skeleton components, features characteristic for cancer cells.