The ε-Isoform of PKC Mediates the Hypertonic Activation of Cation Channels in Confluent Monolayers of Rat Hepatocytes

We were interested whether PKCα, δ, ε or ζ is the isoform actually employed in the activation of hypertonicity-induced cation channels (HICCs) in primary cultures of rat hepatocytes. Quantitative SDS-page and Western-blot experiments revealed that PKCα, δ and ε were stimulated by Indolactam V (as a DAG substitute for activation of c and nPKCs) but that only PKCδ and ε did respond to hypertonic stress. Furthermore, chelation of intracellular Ca++ by BAPTAAM did not alter HICC activation in cable-analysis experiments whereas Indolactam V as well as V8 (an Indolactam derivative specific for PKCδ and ε) activated HICC currents under isotonic conditions. Finally, by use of Rottlerin (as an inhibitor exhibiting a slight preference for PKCδ over ε) PKCε could be identified as the most likely isoform responsible for the activation of the HICC. Introduction In addition to the mere maintenance of homeostasis, the mechanisms of cell volume regulation are employed in a variety of most relevant physiological processes. Among these is the locomotion of cells, the coordination of apical and basal-lateral transport in epithelia, the triggering of cell metabolism and gene expression and, most notably, the control of proliferation as well as apoptosis [1-5]. Whenever analyzed in a quantitative fashion, hypertonicity-induced cation channels (HICCs) were found to be the main mechanism of the regulatory volume increase (RVI) of a shrunken cell (see [6, 7] for review). This also holds for confluent monolayers of rat hepatocytes, from which a relative contribution of the HICC, NHE1 and NKCC1 to hypertonicity-induced Na+ import and RVI of 4: 1: 1was reported [8]. In this system, HICC activation has recently been shown to employ focal adhesion signalling, G proteins, PLC, PKC and finally the exocytotic insertion of channels into the plasma membrane [9, 10]. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 /2 01 7 12 :2 7: 55 A M