Role of Calcium Signaling in Endothelial Barrier Function

Intact endothelia lining the vasculature play a crucial role in tissue homeostasis and organ function. Various blood borne and tissue released mediators influence endothelial barrier function under physiological conditions and become critical under common pathologies such as inflammation and sepsis, known to be associated with increased vascular leakage. Prominent inflamatory mediators such as Thrombin and Histamine transiently disrupt the endothelial barrier via activation of G-protein coupled receptors (GPCRs), while other GPCR agonists, including Sphingosine-1-phosphate (S1P), enhance endothelial barrier function.The barrier disruptive activities of Thrombin and Histamine were repeatedly proposed to be associated with these agonists ability to increase intracellular Calcium (Ca2+) concentrations ([Ca2+]i) via activation of Gq-associated signaling involving phospholipase C (PLC) activation, production of inositol-1,4,5 trisphosphate (IP3), Ca2+ entry through store-operated calcium channels (SOCs) and initiation of Ca2+-dependent endothelial contractility through myosin light chain kinase (MLCK) activation. Here, we use Electric Cell-Substrate Impedance Sensing (ECIS) to challenge this view. We determined barrier function upon stimulation with various barrier modulating agonists in primary human dermal microvascular endothelial cells (HDMECs). We noted obvious discrepancies in the effective agonist concentrations able to evoke either detectable changes in [Ca2+]i or in barrier function which pointed to a complex role of Ca2+ signaling, if any, in endothelial barrier regulation. Absent or inconsistent [Ca2+]i transients under conditions of increased cellular confluency further challenge a role of Ca2+-mediated signaling in receptor-mediated disruption of barrier integrity. A thorough pharmacological examination revealed that different SOC inhibitors (e.g. lanthanides, 2-APB, BTP2) completely abrogated store-operated calcium entry (SOCE) while having no effect on receptor-mediated disruption of endothelial barrier function thus suugesting that SOCE is not required for endothelial barrier regulation.