Bradykinin increases permeability by calcium and 5-lipoxygenase in the ECV304/C6 cell culture model of the blood-brain barrier

The blood-brain barrier (BBB) was modelled in this study using ECV304 cells in co-culture with rat C6 glioma cells, which resulted in elevated transendothelial electrical resistance (TEER). The inflammatory mediator bradykinin (1 μM) was studied and found to induce a fall in TEER; the link between this change and intracellular free calcium concentration ([Ca²⁺]_i) was then examined. 1 μM bradykinin produced a peak-plateau increase in [Ca²⁺]_i. The peak showed desensitization and was dose dependent (over 0.1 nM to 1 μM). The [Ca²⁺]_i increase was blocked by the B₂ antagonist HOE 140 (1 μM) without effect from a B₁ agonist and antagonist. The plateau response was abolished in Ca²⁺-free solution containing 2 mM EDTA, and also by the Ca²⁺ channel blockers lanthanum, La³⁺ (10 μM), and SKF 96365 (100 μM). The store Ca²⁺ATPase inhibitor thapsigargin (1 μM) abolished the peak response. The putative phospholipase C inhibitors, U73122 (20 μM) and ETH-18-OCH₃ (100 μM), unexpectedly increased [Ca²⁺]_i; after their application, bradykinin was ineffective. Agents without effect on Ca²⁺ responses to bradykinin included the phospholipase A₂ (PLA₂) inhibitor aristolochic acid (0.5 mM), cyclooxygenase inhibitor indomethacin (100 μM), 5-lipoxygenase inhibitor nordihydroguaiaretic acid, NDGA (100 μM), calphostin C (0.5 μM), L-NAME (1 mM) and nifedipine (10 μM). The fall in TEER from bradykinin was blocked by HOE 140, U73122 and thapsigargin combined with La³⁺, and also by aristolochic acid and NDGA, but not indomethacin, calphostin C or L-NAME. U73122 increased TEER while ETH-18-OCH₃ reduced it. Thus bradykinin reduced TEER through B₂ receptor-linked release of Ca²⁺ from thapsigargin-sensitive stores, leading to activation of PLA₂ and metabolism of arachidonic acid by 5-lipoxygenase.