Ca2+ signalling and PKC alpha activate increased endothelial permeability by disassembly of VE-cadherin junctions

1. The role of intracellular Ca2+ mobilization in the mechanism of increase d endothelial permeability was studied. Human umbilical vein endothelial ce lls (HUVECs) were exposed to thapsigargin or thrombin at concentrations tha t resulted in similar increases in intracellular Ca2+ concentration ([Ca2+] (i)). The rise in [Ca2+](i) in both cases was due to release of Ca2+ from i ntracellular stores and influx of extracellular Ca2+. 2. Both agents decreased endothelial cell monolayer electrical resistance ( a measure of endothelial cell shape change) and increased transendothelial I-125-albumin permeability. Thapsigargin induced activation of PKC alpha an d discontinuities in VE-cadherin junctions without formation of actin stres s fibres. Thrombin also induced PKC alpha activation and similar alteration s in VE-cadherin junctions, but in association with actin stress fibre form ation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin ligh t chain (MLC20), whereas thrombin induced MLC20 phosphorylation consistent with formation of actin stress filters. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin juncti ons and the decrease in transendothelial electrical resistance caused by bo th agents. Thus, the increased [Ca2+](i) elicited by thapsigargin and throm bin may activate a calphostin C-sensitive PKC pathway that signals VE-cadhe rin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca2+ signalling and activation of PK C alpha in mediating the disruption of VE-cadherin junctions, and thereby i n the mechanism of increased endothelial permeability.