All rats were kept under conditions that conformed to the regulations of the Regierung von Oberbayern and all experiments were performed in accordance with relevant guidelines and regulations. (B) Endothelial cells display vinculin staining under resting conditions while the staining along cell borders is usually disturbed after stimulation with histamine (arrow). (C) Overlay of F-actin and vinculin staining (n?=?4 independent experiments). (D) Co-localization between vinculin and actin, determined by Pearsons correlation coefficient, appears to be slightly reduced due to histamine treatment (n?=?4 independent experiments). Discussion Histamine induces a transient decrease in barrier function accompanied by increased 18-catenin staining along AJs The signaling mechanisms underlying acute endothelial barrier breakdown under inflammatory conditions are not entirely clear. Histamine and thrombin induce acute break-down of the endothelial barrier. However, thrombin is not a typical inflammatory mediator since it regulates vascular permeability during the coagulation phase after injury39 and in many studies macrovascular endothelial cells were used, which are good models for investigating adaptations to sheer stress but not the inflammatory response, which occurs exclusively in postcapillary venules3. Therefore, re-evaluation of all findings using microvascular endothelium is necessary. Histamine impaired the integrity of the endothelial barrier and and postcapillary venules of rat mesentery only because intact mesenteric venules do not respond to thrombin37,38. Consistent with the literature42, our data showed that histamine and thrombin disrupted the endothelial barrier transiently and caused reduced TER and intercellular gap formation. At the same time, both thrombin and histamine enhanced staining with the conformation-sensitive antibody targeting the alpha 18-subunit of catenin which has been proposed to correlate with increased tension at AJs (Fig.?8). Increased tension may result from RhoA-dependent up-regulation of actomyosin contraction27 which may trigger relocalization of vinculin to AJs from FAJs16. Therefore, we correlated the different events during endothelial barrier breakdown with immunostaining for vinculin, which is known to be a mechanotransducer with the ability to stabilize adhesion under force16,44C46. Interestingly, we observed that vinculin was localized at cell junctions under resting conditions both in HDMECs and endothelial cells of postcapillary venules whereas Rho kinase inhibition was not. These observations suggest that inactivation of Rac1 rather than stimulation of the RhoA/Rho kinase pathway was the primary mechanism underlying TNF–mediated permeability increase. Surprisingly, our results of the present study showed no differences in Rac1 activity or in cAMP concentrations after treatment with histamine. Therefore, we conclude that in contrast to the situation in sepsis, where LPS and TNF- are relevant, alterations of cAMP levels as well as Rac1 activity are less important in anaphylaxis. Rather, acute inflammation appears to be primarily dependent on Ca2+ and activation of RhoA. Ca2+ signaling is required for RhoA activation, AJ reorganization and barrier dysfunction Permeability mediators, including histamine, act on Gq-coupled GPCRs leading the activation of PLC-? and the rapid mobilization of Ca2+, which promotes MLC phosphorylation and the stimulation of its contractile activity27. RhoA can be activated independently by H1R coupled to Gq/1128. H1R also leads to an Parecoxib increase of intracellular Ca2+ concentration. As reported Parecoxib previousley28, we found that H1 receptor is required for histamine to induce permeability (Fig.?8). Our results revealed that both histamine and thrombin lead to a rapid Ca2+-influx accompanied by parallel reduction of TER. Chelation of Ca2+ by BAPTA-AM similar to inhibition of ROCK blocked the effects of histamine and thrombin on barrier function and AJ reorganization. Moreover, BAPTA-AM prevented activation of RhoA and similar to inhibition of ROCK blunted 18-catenin staining along junctions. These results indicate that Ca2+ -mediated RhoA activation is critical for endothelial barrier regulation (Fig.?8). The pivotal role of RhoA has been exhibited previously27, however, in this PVR study the authors concluded that RhoA was activated primarily impartial of Ca2+. In addition, we observed Parecoxib that stress fiber formation in response to histamine was abrogated by chelation of Ca2+ but not by inhibition of Rho kinase. This supports the notion that in response to histamine stress fiber formation can be induced by Ca2+ in a manner impartial of Rho kinase, most likely via MLCK50, and suggests that stress fibers are not directly involved in tension formation at AJ and endothelial barrier breakdown (Fig.?8). Conclusion Therefore, we conclude that Ca2+ via RhoA/ROCK activation is critical for histamine-induced barrier disruption which correlated with tension-associated 18-catenin staining along endothelial AJ. These data suggests that the patterns of signaling mechanisms engaged in acute inflammatory barrier disruption differ to more delayed responses as seen in.