Cross-talk between PKA and Akt protects endothelial cells from apoptosis in the late ischemic preconditioning.
OBJECTIVE
The aim of this study was to explore the molecular mechanisms involved in late preconditioning-induced cell protection in endothelial cells.


RESULTS
Preconditioning (PC) was induced by exposing bovine aortic endothelial cells (BAECs) to 3 cycles of 15 minutes of hypoxia followed by 15 minutes of reoxygenation. A 12-hour period of hypoxia induced cell death in 60% of BAECs (48+/-5% apoptosis, 12+/-4% necrosis). Early and late PC decreased hypoxia-induced apoptotic (25+/-5% and 28+/-4%, respectively) and necrotic (6+/-3%, and 8+/-2%, respectively) cell death. Consistently, hypoxia-induced caspase-3 cleavage was reduced by PC. Pretreatment with H89 (protein kinase A [PKA] inhibitor), LY294002 (phosphatidyl-inositol-3-kinase [PI3K] inhibitor), and N-acetyl-cysteine (antioxidant) abrogated late PC-induced cell protection, whereas inhibition of protein kinase C by Go6983, and of nitric oxide synthesis by L-NAME,1400W and bovine eNOS siRNA did not. In addition, in early and late PC, PKA physically interacted with the phosphorylated form of Akt, suggesting that PKA is required for Akt phosphorylation. Expression of PKA and Akt dominant negative mutants inhibited ischemic late PC-induced protection, indicating that these kinases play a key role in late PC-mediated cell protection.


CONCLUSIONS
Late ischemic PC protects BAECs against hypoxia through PKA- and PI3K-dependent activation of Akt.
Bellis
Alessandro
A
Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche. Università FEDERICO II, Napoli, Via S. Pansini n.5, 80131 Napoli, Italy.
Castaldo
Diletta
D
Trimarco
Valentina
V
Monti
Maria Gaia
MG
Chivasso
Pierpaolo
P
Sadoshima
Junichi
J
Trimarco
Bruno
B
Morisco
Carmine
C
D000818:Animals; D001011:Aorta / Q000473:pathology; D017209:Apoptosis / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology*; D015151:Immunoblotting; D007511:Ischemia / Q000378:metabolism / Q000473:pathology*; D019194:Ischemic Preconditioning; D009569:Nitric Oxide / Q000096:biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D020239:Receptor Cross-Talk / Q000502:physiology*; D013997:Time Factors
D009569:Nitric Oxide; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases