Effect of protein kinase C activation on intracellular Ca2+ signaling and integrity of intestinal epithelial cells

Protein kinase C (PKC) activation and increases in cytosolic Ca2+ cause intestinal injury. Since PKC activation can alter Ca2(+) homeostasis and increase Ca2+ levels, we examined the effects of PKC activation on intestinal cellular integrity and the role of Ca2+ signaling in this response. The epithelial cell line, IEC-18 was incubated with the PKC activator phorbol myristate acetate (PMA; 0.1-1.0 mu M). In some experiments, cells were incubated in Ca2+-free medium. PMA treatment produced a concentration-dependent increase in cell injury and PKC activity. This response was attenuated by addition of the pan-specific PKC inhibitor, GF 109203X. Furthermore, cell viability was maintained in cells preincubated with PKC isoform-specific inhibitors to PKC alpha, PKC delta and PKC epsilon. Cell injury was also reduced if cells were incubated in Ca2+-free medium or in the presence of the Ca2+ channel antagonist, verapamil or the intracellular chelator BAPTA-AM. PMA, but not the inactive phorbol ester, 4 alpha PMA, induced a dose-dependent increase in cellular Ca2+ that was characterized by a rapid, transient spike followed by a tonic plateau phase which approximated control levels. These responses were eliminated by the addition of BAPTA-AM. Furthermore the increase in the Ca2+ spike was reduced or eliminated by co-incubation with the PKC delta antagonist, rottlerin. Inhibition of PKCa or PKCe was less effective or ineffective in this regard. These data suggest that PKC activation via PMA challenge affects the integrity of rat intestinal epithelial cells. PKC delta, but not PKC epsilon or PKC alpha activation appears to mediate this effect via an increase in cellular Ca2+. (c) 2005 Elsevier B.V All rights reserved.