Activation of protein kinase C isozymes protects LLCPK1 cells from H2O2 induced necrotic cell death.
OBJECTIVE
We have previously reported that ischemia/reperfusion injury (IRI) to the kidney leads to induced expression of RACK1 and changes in the level of expression and subcellular distribution of PKC isozymes alpha, betaII and zeta. In order to further define the role of PKC isozymes in IRI we investigated the effect of activation or inhibition of the isozymes on cytotoxicity mediated by H(2)O(2) in LLCPK(1) cells.


METHODS
Cytotoxicity was analyzed by Trypan blue assay and LDH release assay. Translocation of PKC isozymes postinjury in LLCPK1 cells was analyzed by immunostaining and Western blot analysis.


RESULTS
Western blot analysis showed that the expression of PKC-alpha was up-regulated in a triphasic pattern with the initial induction within the first 10 min of injury followed by higher levels of expression at 2 and 24 h postinjury. The expression of PKC-zeta was highly induced within the first 15 min of injury but its expression was down-regulated to that of normal levels by 30 min postinjury. Immunocytochemistry showed that both PKC-alpha and PKC-zeta translocated to the nucleus and perinuclear region during H(2)O(2) treatment. Following injury, PKC-alpha expression was localized to the nuclear membrane at earlier time points but a translocation to the nucleus occurred at later time points. PKC-zeta translocated to nucleus at 30 minutes post injury and relocated back to the nuclear membrane at later time points.


CONCLUSIONS
These data suggest that activation of PKC-alpha and PKC-zeta is involved in the H(2)O(2) induced injury of LLCPK1 cells.
Polosukhina
Dina
D
Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebr. 68198-4575 USA. bpadanilam@unmc.edu
Singaravelu
Kurinji
K
Padanilam
Babu J
BJ
D000818:Animals; D015153:Blotting, Western; D016923:Cell Death; D019610:Cytoprotection; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000633:toxicity; D007527:Isoenzymes / Q000378:metabolism; D018374:LLC-PK1 Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009336:Necrosis; D016877:Oxidants / Q000633:toxicity; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D016877:Oxidants; D006861:Hydrogen Peroxide; D011493:Protein Kinase C