Disruption of the endothelial barrier by pro‐inflammatory cytokines is a complication of multiple diseases, including sepsis that can result in tissue edema. To assess the effect of TNFα on native endothelial permeability, we customized an Ussing chamber to record electrophysiological measurements of ex vivo mesenteric mouse venules or arterioles that were treated with either DMSO vehicle control or 50ng/ml TNFα for 30 minutes. Venules showed a TNFα‐dependent decrease in transendothelial electrical resistance (TEER), whereas arterioles did not. An in situ microvessel perfusion system also demonstrated preferential increases in fluorescein permeability in TNFα treated venules versus arterioles. Human umbilical and saphenous vein (HUVEC and HSaVEC) cells cultured on Transwell permeable supports showed a comparable decrease in TEER in response to TNFα; cultured aortic and coronary arterial (HAoEC and HCoEC) endothelial cells did not. At tight junctions, we found that in addition to claudin‐1 and claudin‐5, venous endothelial cells also expressed claudin‐11. Upon addition of TNFα, venous claudin protein expression was significantly reduced. To investigate molecular mechanisms underlying the differential effect of TNFα on venous and arterial cells, we targeted pannexin 1 (PANX1) channel activity using the antagonists mefloquine and spironolactone in vitro and in venules from conditional PANX1 knockout mice and found that in all cases TNFα‐induced permeability was eliminated. Further analysis of purinergic signaling in venous endothelium revealed that the ectonucleotidase CD39 is expressed at higher levels in veins than arteries and that addition of the CD39‐antagonist ARL67156 abolished TNFα‐induced permeability in venules from wildtype mice. Coupled with expression of CD73, this result suggests that ATP released by PANX1 is metabolized to adenosine as an autocrine signal produced in response to TNFα. Consistent with adenosine signaling, TNFα‐induced permeability was impaired in venules isolated from wildtype mice in the presence of A2A and A2 antagonists as well as in venules from A2A knockout mice. Our results demonstrate that targeting the purinergic signaling pathway mitigates TNFα‐induced permeability in venous cells. These findings offer multiple possibilities for pharmacological interventions aimed at preventing and treating increased vascular permeability during inflammation.Support or Funding InformationR01 AA025854This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.