Preconditioning of bovine endothelial cells: The protective effect is mediated by an adenosine A₂ receptor through a protein kinase C signaling pathway

We tested the hypothesis that anoxic preconditioning could protect coronary endothelial cells against anoxic and reoxygenation injury and that this preconditioning effect could be mediated by an adenosine A₂ receptor via the protein kinase C (PKC) pathway. Cells were preconditioned with 10- minute anoxia and 10-minute reoxygenation and were then subjected to anoxia for 60 minutes, followed by 120 minutes of reoxygenation. In some groups, the preconditioning effect was prevented by 8-sulfophenyltheophylline (SPT [50 μmol/L], a nonselective adenosine receptor antagonist) or calphostin C (100 nmol/L, a PKC inhibitor). In other groups, 2-p-(2- carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680 [20 nmol/L], an adenosine A₂ receptor agonist), R-(-)-N⁶-(2-phenylisopropyl)- adenosine (R-PIA [50 nmol/L], an adenosine A₁ receptor agonist), or 4β- phorbol 12-myristate 13-acetate (PMA) [100 nmol/L], a PKC activator) was given as a pretreatment to mimic the preconditioning effect. Endothelial cells were also pretreated with 100 nmol/L calphostin C to confirm whether inhibition of PKC can block the effects of adenosine A₂ receptor activation by CGS-21680 on anoxia and reoxygenation injury. Preconditioning reduced LDH release, increased adenosine release, promoted translocation of PKC from cytosol to membrane, increased cell viability, and preserved ATP content and cell morphology. Pretreatment with either CGS-21680 or PMA resulted in protection similar to that seen with anoxic preconditioning. The protection was totally abolished by SPT or calphostin C. The results suggest that (1) preconditioning protects coronary endothelial cells against anoxia and reoxygenation injury, (2) the protection is probably mediated by activation of adenosine A₂ receptors through the PKC pathway, and (3) the preservation of endothelial cells may be one of the mechanisms of myocardial preconditioning.