Background: 

Recently we showed that hypoxia induces an endogenous anti-apoptotic mechanism in human endothelial cells. Here we tested the hypothesis whether the protective effect is due to a release of adenosin-5'-triphosphat (ATP) during transient hypoxia.

Methods and Results: 

In cultured human vein endothelial cells (HUVEC) apoptosis was induced by serum-starvation for 16 hrs. Subsequently cells were exposed to 2 hrs of hypoxia followed by 24 hrs of reoxygenation. Exposure to transient hypoxia reduced caspase 3 activity by 352% (Western blot analysis) and decreased apoptosis by 223% (annexin V staining, FACS) compared to normoxic control (P<0.05, n=3 for all further parameters). During hypoxia a 2.70.7-fold increase of ATP was observed in the supernatant (luciferin/luciferase assay). The ATP release was accompanied by a 1.90.2-fold increase of ERK2-phosphorylation. Inhibition of the MEK/ERK pathway by using PD98059 (10mM), a specific MEK1-inhibitor, abrogated the reduction of caspase 3 activity and apoptosis. Enhanced degradation of ATP during hypoxia in presence of apyrase/adenosine deaminase (each 1 U/ml) inhibited ERK2-phosphorylation and increased caspase 3 activity. In contrast, addition of the ectonucleotidase-inhibitor ARL 67156 (100mM) or exogenous addition of ATP (10 mM) during hypoxia resulted in a significant increase of ERK2-phosphorylation and a reduction of caspase 3 activity, respectively. Suramin, a panspecific P2-receptor-antagonist, abrogated the anti-apoptotic effect of transient hypoxia. The addition of the stable ATP-analogue ATPgS, as well as the addition of the P2Y2-agonist UTP showed the same protective effect as the inhibition of the ectonucleotidase. However, the addition of the P2Y1-agonist MRS 2365 or the addition of the P2X-agonist aßATP had no effect on caspase 3 activity.

Conclusion: 

In human endothelial cells hypoxia induces a release of ATP which activates an autocrine survival mechanism via a P2Y2 receptor-mediated MEK/ERK signaling.