Hypoxia and reoxygenation-induced oxidant production increase in microvascular endothelial cells depends on connexin40.

Connexins (Cx) are recognized as structural constituents of gap-junctional intercellular communication (GJIC). However, their function may extend beyond facilitating the exchange of metabolites and electrical signals between cells. In this study we asked if increased production of reactive oxygen species (ROS) in microvascular endothelial cells challenged by hypoxia/reoxygenation (H/R) requires Cx40, independent of GJIC. Because we showed that this ROS increase depends on NADPH oxidase, we also asked if Cx40 function (i.e., Cx40-dependent reduction in interendothelial electrical coupling after H/R) requires NADPH oxidase. ROS increase was assessed in confluent monolayers of cultured endothelial cells derived from skeletal muscle blood vessels of wild-type (WT) and Cx40(-/-) mice and in monolayers of GJIC-deficient SKHep1 cells overexpressing GFP-tagged Cx40. Electrical coupling was assessed in WT cells and in cells lacking the NADPH oxidase subunit gp91phox or p47phox. H/R elicited a 70-80% ROS increase in WT but not in Cx40(-/-) cells. The increase was not affected by the gap junction blocker 18alpha-glycyrrhetinic acid or by preventing the cells from establishing cell-to-cell contact. H/R increased ROS in SKHep1 cells expressing Cx40-GFP, but not in cells expressing the control vector. Finally, H/R reduced electrical coupling in WT and gp91phox(-/-) but not in p47phox(-/-) cells. Our data indicate that (i) the H/R-induced ROS increase in microvascular endothelial cells requires Cx40, independent of its role in GJIC, and (ii) p47phox rather than NADPH oxidase-derived ROS affects modulation of intercellular coupling. Together, the results raise an intriguing possibility that H/R-induced signaling in endothelial cells involves a cross-talk between Cx40 and NADPH oxidase.