Spontaneous injury to human umbilical vein endothelial cells increases during in vitro culture and is blocked by protein kinase activation.
BACKGROUND
The in vitro culture of human umbilical vein endothelial (Huve) cells alters sensitivity to cell injury. Late passage Huve cells in basal media have an increased susceptibility to spontaneous injury and this type of cell injury is blocked by phorbol myristate acetate (PMA).


METHODS
Protein kinase activation and alterations in free radical generation and defense were investigated as potential mechanisms for the increased spontaneous injury of late passage Huve cells in basal media and for the PMA-mediated protective response.


RESULTS
The spontaneous injury of late passage Huve cells in basal media was blocked by PMA via a protein kinase-dependent mechanism. Huve cells cultured in vitro for several passages had increased basal particulate fraction protein kinase activity and increased activity in response to PMA treatment as compared with first passage cells. The spontaneous injury was not reduced by addition of free radical scavengers or iron chelators. The increased spontaneous injury in late passage Huve cells, and the protective effects of PMA treatment were not explained by alterations in the endogenous antioxidant defense system. PMA-mediated protection against spontaneous injury was blocked by staurosporine, a protein kinase inhibitor, but not by addition of various free radical scavengers or chelators.


CONCLUSIONS
These data suggest that the cellular changes mediated by protein kinase are more important than free radical metabolism or antioxidant defense as an explanation for the increased spontaneous injury of late passage Huve cells in basal media and for the ability of PMA to block this type of spontaneous cell injury.
Department of Pathology, University of Michigan Medical School, Ann Arbor.
C G
CG
Taylor
M K
MK
Dame
H S
HS
Murphy
P A
PA
Ward
J
J
Varani
D000470:Alkaloids / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000293:injuries*; D004789:Enzyme Activation; D016166:Free Radical Scavengers; D005978:Glutathione / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000494:pharmacology; D006801:Humans; D007502:Iron Chelating Agents / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013113:Spin Labels; D019311:Staurosporine; D013482:Superoxide Dismutase / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014471:Umbilical Veins
D000470:Alkaloids; D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D007502:Iron Chelating Agents; D013113:Spin Labels; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D011494:Protein Kinases; D011493:Protein Kinase C; D005978:Glutathione; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate