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 muM) 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 muM bradykinin produced a peak-plateau increase in [Ca2+](i). The peak showed desensitization and was dose dependent (over 0.1 nM to 1 muM). The [Ca2+](i) increase was blocked by the B-2 antagonist HOE 140 (1 muM) without effect from a B-1 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 muM), and SKF 96365 (100 muM). The store Ca(2+)ATPase inhibitor thapsigargin (1 muM) abolished the peak response. The putative phospholipase C inhibitors, U73122 (20 muM) and ETH-18-OCH3 (100 muM), unexpectedly increased [Ca2+](i); after their application, bradykinin was ineffective. Agents without effect on Ca2+ responses to bradykinin included the phospholipase A(2) (PLA(2)) inhibitor aristolochic acid (0.5 mM), cyclooxygenase inhibitor indomethacin (100 muM), 5-lipoxygenase inhibitor nordihydroguaiaretic acid, NDGA (100 P M), calphostin C (0.5 muM), L-NAME (I mM) and nifedipine (10 muM). 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 B-2 receptor-linked release of Ca2+ from thapsigargin-sensitive stores, leading to activation of PLA(2) and metabolism of arachidonic acid by 5-lipoxygenase. (C) 2002 Elsevier Science B.V. All rights reserved.