Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood-brain barrier compromise.
BACKGROUND
Brain endothelial cells form the blood-brain barrier (BBB) that regulates solute and macromolecule flux in and out of the brain, leukocyte migration, and maintains the homeostasis of the central nervous system. BBB dysfunction is associated with disruption of tight junctions (TJ) in the brain endothelium. We propose that alcohol abuse may impair BBB permeability through TJ modification.


METHODS
Primary cultured bovine brain microvascular endothelial cells (BBMEC) were treated with 50 mM ethanol (EtOH), and monolayer tightness was assessed by measurement of transendothelial electrical resistance (TEER). Changes in TEER were correlated with alterations in TJ protein distribution [occludin, zonula occludens-1 (ZO-1), claudin-5] using immunofluorescence (IF). Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot. EtOH-induced changes in monocyte migration across in vitro BBB constructs were also examined.


RESULTS
EtOH induced a decrease in TEER of BBMEC monolayers that was reversed by EtOH withdrawal. Treatment of BBMEC with EtOH or its metabolite, acetaldehyde, prior to monocyte application resulted in a 2-fold increase in monocyte migration across the BBB. IF demonstrated decrease in claudin-5 staining, occludin translocation from cell borders to cytoplasm and gap formation in EtOH-treated BBMEC monolayer. These changes paralleled significant increase in phosphorylation of MLC, occludin and claudin-5. EtOH-treated BBMEC showed reduction of total occludin and claudin-5 without changes in ZO-1 or MLC. TEER decrease, changes in occludin/claudin staining, increase in MLC, occludin and claudin-5 phosphorylation and enhanced monocyte migration across the BBB were all reversed by inhibition of MLCK. Inhibition of EtOH metabolism in BBMEC also reversed these events.


CONCLUSIONS
These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.
Haorah
James
J
Center for Neurovirology and Neurodegenerative Disorders, Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Heilman
David
D
Knipe
Bryan
B
Chrastil
Jesse
J
Leibhart
Jessica
J
Ghorpade
Anuja
A
Miller
Donald W
DW
Persidsky
Yuri
Y
D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000201:enzymology / Q000503:physiopathology*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000493:pharmacokinetics / Q000494:pharmacology*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000187:drug effects / Q000378:metabolism*; D019108:Tight Junctions / Q000201:enzymology / Q000378:metabolism*
D008565:Membrane Proteins; D000431:Ethanol; D009219:Myosin-Light-Chain Kinase