Abstract 247: ATP is released during Hypoxia/Reoxygenation via Connexin 43 Hemichannels and protects Endothelial Cells from Apoptosis
Introduction: Recently, we have shown that hypoxia/reoxygenation (H/R) causes release of ATP from endothelial cells (EC). Here we analyzed the ATP release mechanism and hypothesize that ATP passes through the cell membrane via connexin 43 (Cx43) hemichannels and protects endothelial cells against apoptosis during H/R.
Methods and Results: In cultured human umbilical vein endothelial cells apoptosis was induced by serum deprivation. Subsequently, cells were exposed to 2 hrs of hypoxia (Po2<10mmHg) followed by 24 hrs of reoxygenation (Po2=140 mmHg). Control cells were exposed to normoxia for 26 hrs. Induction of apoptosis was confirmed under normoxic conditions by analyzing caspase 3 activation (western blot) and annexin V staining (FACS analysis). Hypoxia significantly reduced caspase 3 activation by 35±7% and apoptosis by 22±3% compared to the normoxic controls (n≥3; P<0.05, for all further parameters). The reduction of apoptosis was accompanied by a 1.9±0.2 -fold increase of ERK-2 phosphorylation. Inhibition of MEK/ERK by PD 98059 (10μM), a specific MEK1 inhibitor, abolished the hypoxia-induced effect on caspase 3 activity and apoptosis. Hypoxia induced a transient 1.6±0.3 - fold increase of ATP concentration in the supernatant (luciferase assay). Enhanced degradation of ATP during hypoxia by apyrase (1 U/ml) / adenosine desaminase (1 U/ml) abrogated the hypoxia-induced reduction of caspase 3 activity. Downregulation of Cx43 by siRNA silencing abolished ATP release, ERK-2 phosphorylation as well as the hypoxia-induced cell survival.
Conclusion: The data of the present study show that hypoxia induces ATP release through Cx43 hemichannels. This ATP activates an antiapoptotic signaling, involving MEK/ERK pathway, and prevents EC from apoptotic cell death.