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 Ca(2+) cause intestinal injury. Since PKC activation can alter Ca(2+) homeostasis and increase Ca(2+) levels, we examined the effects of PKC activation on intestinal cellular integrity and the role of Ca(2+) signaling in this response. The epithelial cell line, IEC-18 was incubated with the PKC activator phorbol myristate acetate (PMA; 0.1-1.0 microM). In some experiments, cells were incubated in Ca(2+)-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 PKCalpha, PKCdelta and PKCepsilon. Cell injury was also reduced if cells were incubated in Ca(2+)-free medium or in the presence of the Ca(2+) channel antagonist, verapamil or the intracellular chelator BAPTA-AM. PMA, but not the inactive phorbol ester, 4alphaPMA, induced a dose-dependent increase in cellular Ca(2+) 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 Ca(2+) spike was reduced or eliminated by co-incubation with the PKCdelta antagonist, rottlerin. Inhibition of PKCalpha or PKCepsilon 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. PKCdelta, but not PKCepsilon or PKCalpha activation appears to mediate this effect via an increase in cellular Ca(2+).

The role of PKCdelta and PKCepsilon in the neonatal rat colon in response to hypoxia challenge.

Previous studies have determined that, in response to bacterial endotoxin, the colonic mucosa of the 10-d-old neonatal rat was more susceptible to injury than was the colon of the 25-d-old mature animal. Furthermore, it is known that certain isoforms of protein kinase C (PKC), specifically PKCdelta and PKCepsilon, mediate intestinal inflammatory responses to specific challenges. Therefore, in the present study, we have examined the association between the activation of these PKC isoforms and the enhanced susceptibility to hypoxia-induced challenge. In response to exposure to a hypoxic environment (14% O2/86% N2, 30 min), the degree of inflammation and tissue damage was significantly greater in 10- than in 25-d-old rats. The injury in 10-d-old rats was associated with activation of PKCdelta and PKCepsilon as estimated by translocation of the isoform from cytosolic to membrane fraction of the tissue lysate. There was no activation of either isoform in colons from 25-d-old rats after hypoxia. Pretreatment of 10-d-old rats with epidermal growth factor (EGF) (10 microg/kg) but not 16,16 dimethyl prostaglandin E2 (2 microg/kg) significantly reduced the extent of colonic injury, whereas neither agent was able to exert significant protection of the colonic mucosa of 25-d-old rats. PKC activation associated with hypoxia was not evident after EGF treatment in 10-d-old rats. In 25-d-old rats, prostaglandin E2 treatment was linked with an activation of PKCepsilon only. In conclusion, these data suggest that activation of PKCdelta and PKCepsilon is associated with the enhanced susceptibility to injury evident in suckling neonatal rat colon. EGF-mediated protection of the colon in these animals results in a removal of this PKC activation.