Abstract 5506: ATP Released During Hypoxia/Reoxygenation Activates A P2Y Receptor Mediated Survival Mechanism In Human Endothelial Cells
Introduction: Recently we have shown that ATP passes the endothelial cell membrane via Connexin43 hemichannels during hypoxia/reoxygenation (H/R). Here we hypothesized that extracellular ATP protects endothelial cells (EC) against ongoing apoptotic cell death during H/R by activating an autocrine signaling mechanism mediated by purine receptors.
Methods and Results: In cultured human umbilical vein EC a robust apoptosis was induced by serum deprivation. Subsequently, cells were exposed to 2 hrs of hypoxia (Po2<10mmHg) followed by 24 hrs of reoxygenation. Hypoxia reduced caspase 3 activation by 35±7% (western blot, immunocytochemistry) and apoptotic cell death by 22±3% (annexin V staining) compared to the normoxic controls (n ≥ 3; P < 0.05, for all further parameters). During hypoxia a 2.6±0.7 - fold increase of ATP concentration in the supernatant (luciferin-luciferase assay) was observed. The release of ATP was accompanied by a 1.9±0.2 -fold increase of ERK-2 phosphorylation. Inhibition of MEK/ERK by PD 98059 (10μM, MEK1 inhibitor) abolished the hypoxia-induced reduction of caspase 3 activity and apoptosis. Downregulation of Cx43 hemichannels by siRNA silencing abolished ATP release, ERK 2 phosphorylation and the hypoxia induced cell survival. However, by adding ATP (10μM) exogenously the ERK 2 phosphorylation as well as the reduction of caspase 3 activity could even be potentiated. Enhanced degradation of ATP by using apyrase (1U/ml) in contrast decreased the ERK 2 phosphorylation and aggravated caspase 3 activity. The protective effect of extracellular ATP could be mimiced by using the non - hydrolyzable P2Y receptor agonist ATPgammaS (10μM). The addition of suramin (100μM), a P2Y receptor antagonist abrogated the hypoxia induced reduction of caspase 3 activity. Stimulation of adenosine receptors by NECA (1 μM) or degradation of adenosine by adenosine desaminase (1U/ml) during hypoxia had no effect on caspase 3 activity.
Conclusion: The data of the present study show that hypoxia induces ATP release via Cx43 hemichannels in EC. The extracellular ATP protects EC against ongoing apoptotic cell death by activating an autocrine survival mechanism. This protective mechanism is due to a P2Y receptor mediated signaling involving the MEK/ERK pathway.