Extracellular
ATP is an important signaling molecule for vascular adaptation to limited
oxygen availability (
hypoxia). Here, we pursued the contribution of vascular endothelia to extracellular
ATP release under hypoxic conditions.
METHODOLOGY, PRINCIPAL FINDINGS: We gained first insight from studying
ATP release from endothelia (HMEC-1) pre-exposed to
hypoxia. Surprisingly, we found that
ATP release was significantly attenuated following
hypoxia exposure (2%
oxygen, 22+/-3% after 48 h). In contrast, intracellular
ATP was unchanged. Similarly,
lactate-dehydrogenase release into the supernatants was similar between normoxic or hypoxic endothelia, suggesting that differences in lytic
ATP release between normoxia or
hypoxia are minimal. Next, we used pharmacological strategies to study potential mechanisms for endothelial-dependent
ATP release (eg,
verapamil,
dipyridamole, 18-alpha-
glycyrrhetinic acid,
anandamide,
connexin-mimetic
peptides). These studies revealed that endothelial
ATP release occurs--at least in part--through
connexin 43 (
Cx43) hemichannels. A real-time RT-PCR screen of endothelial
connexin expression showed selective repression of
Cx43 transcript and additional studies confirmed time-dependent
Cx43 mRNA, total and
surface protein repression during
hypoxia. In addition,
hypoxia resulted in Cx43-serine368 phosphorylation, which is known to switch
Cx43 hemi-channels from an open to a closed state.
CONCLUSIONS/SIGNIFICANCE: