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 microM) was studied and found to induce a fall in TEER; the link between this change and intracellular free calcium concentration ([Ca(2+)](i)) was then examined. 1 microM bradykinin produced a peak-plateau increase in [Ca(2+)](i). The peak showed desensitization and was dose dependent (over 0.1 nM to 1 microM). The [Ca(2+)](i) increase was blocked by the B(2) antagonist HOE 140 (1 microM) without effect from a B(1) agonist and antagonist. The plateau response was abolished in Ca(2+)-free solution containing 2 mM EDTA, and also by the Ca(2+) channel blockers lanthanum, La(3+) (10 microM), and SKF 96365 (100 microM). The store Ca(2+)ATPase inhibitor thapsigargin (1 microM) abolished the peak response. The putative phospholipase C inhibitors, U73122 (20 microM) and ETH-18-OCH(3) (100 microM), unexpectedly increased [Ca(2+)](i); after their application, bradykinin was ineffective. Agents without effect on Ca(2+) responses to bradykinin included the phospholipase A(2) (PLA(2)) inhibitor aristolochic acid (0.5 mM), cyclooxygenase inhibitor indomethacin (100 microM), 5-lipoxygenase inhibitor nordihydroguaiaretic acid, NDGA (100 microM), calphostin C (0.5 microM), L-NAME (1 mM) and nifedipine (10 microM). The fall in TEER from bradykinin was blocked by HOE 140, U73122 and thapsigargin combined with La(3+), and also by aristolochic acid and NDGA, but not indomethacin, calphostin C or L-NAME. U73122 increased TEER while ETH-18-OCH(3) reduced it. Thus bradykinin reduced TEER through B(2) receptor-linked release of Ca(2+) from thapsigargin-sensitive stores, leading to activation of PLA(2) and metabolism of arachidonic acid by 5-lipoxygenase.
Easton
Alexander S
AS
Centre for Neuroscience Research, School of Biomedical Sciences, King's College London, London SE1 1UL, UK. aeaston@ualberta.ca
Abbott
N Joan
NJ
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000502:physiology; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000493:pharmacokinetics*; D002463:Cell Membrane Permeability / Q000187:drug effects; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005910:Glioma; D006801:Humans; D019001:Nitric Oxide Synthase / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured
D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D016718:Arachidonic Acid; D001094:Arachidonate 5-Lipoxygenase; D019001:Nitric Oxide Synthase; D011493:Protein Kinase C; D001920:Bradykinin; D002118:Calcium