Downregulation of protein kinase cdelta activity enhances endothelial cell adaptation to hypoxia.
BACKGROUND
Although protein kinase C (PKC) has been implicated in ischemic cell death, the role of individual PKC isoenzymes in the response of endothelial cells (ECs) to hypoxia is unknown.


RESULTS
To test the effect of hypoxia on the activity of individual PKC isoenzymes, human ECs were exposed to 95% N(2) with 5% CO(2) for 24 hours. This severe hypoxia reduced PKCdelta specific activity in both human umbilical vein ECs (HUVECs) and a HUVEC-derived EC line (ECVs) significantly (80.5+/-5.7% and 55.5+/-8. 6% of normoxia controls, respectively); the activities of PKCalpha and PKCepsilon were unchanged. The protein levels of PKCalpha, PKCdelta, and PKCepsilon were unchanged by hypoxia. To determine whether PKCdelta downregulation by hypoxia was linked to EC function, ECVs in which PKCdelta was stably overexpressed (PKCdelta-ECs) were exposed to hypoxia. A significant increase in cell death was observed in PKCdelta-ECs compared with controls (5.8+/-0.6% versus 2. 3+/-0.4% at 24 hours, 13.2+/-1.2% versus 4.1+/-0.4% at 48 hours, P<0. 05) during hypoxia. Neither the DNA laddering assay nor TUNEL staining revealed an increase in apoptosis of PKCdelta-ECs exposed to hypoxia, suggesting a hypoxia-induced increase in nonapoptotic cell death of PKCdelta-ECs. Inhibition of NO synthase with N(G)-monomethyl-L-arginine (L-NMMA) affected neither the decline in PKCdelta activity nor the EC death induced by hypoxia.


CONCLUSIONS
PKCdelta activity is decreased by hypoxia by a mechanism that does not involve NO synthase; this downregulation appears to enhance EC survival during hypoxia by decreasing nonapoptotic cell death.
Shizukuda
Y
Y
Cardiovascular Division, Department of Medicine, and the Department of Molecular Pharmacology, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY, USA.
Helisch
A
A
Yokota
R
R
Ware
J A
JA
D000222:Adaptation, Physiological; D017209:Apoptosis / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019323:omega-N-Methylarginine; D019001:Nitric Oxide Synthase; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta