title: TRPC-mediated actin-myosin contraction is critical for BBB disruption following hypoxic stress.
abstract: Hypoxia-induced disruption of the blood-brain barrier (BBB) is the result of many different mechanisms, including alterations to the cytoskeleton. In this study, we identified actin-binding proteins involved in cytoskeletal dynamics with quantitative proteomics and assessed changes in subcellular localization of two proteins involved in actin polymerization [vasodilator-stimulated phosphoprotein (VASP)] and cytoskeleton-plasma membrane cross-linking (moesin). We found significant redistribution of both VASP and moesin to the cytoskeletal and membrane fractions of BBB endothelial cells after 1-h hypoxic stress. We also investigated activation of actin-myosin contraction through assessment of phosphorylated myosin light chain (pMLC) with confocal microscopy. Hypoxia caused a rapid and transient increase in pMLC. Blocking MLC phosphorylation through inhibition of myosin light chain kinase (MLCK) with ML-7 prevented hypoxia-induced BBB disruption and relocalization of the tight junction protein ZO-1. Finally, we implicate the transient receptor potential (TRP)C family of channels in mediating these events since blockade of TRPC channels and the associated calcium influx with SKF-96365 prevents hypoxia-induced permeability changes and the phosphorylation of MLC needed for actin-myosin contraction. These data suggest that hypoxic stress triggers alterations to cytoskeletal structure that contribute to BBB disruption and that calcium influx through TRPC channels contributes to these events.
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: Kali
authors.initials: K
authors.lastname: Hicks
authors.forename: Roger G
authors.initials: RG
authors.lastname: O'Neil
authors.forename: William S
authors.initials: WS
authors.lastname: Dubinsky
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Brown
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D013312:Stress, Physiological; D050052:TRPC Cation Channels / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D000199:Actins; D001381:Azepines; D002121:Calcium Channel Blockers; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008840:Microfilament Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; D010750:Phosphoproteins; D050052:TRPC Cation Channels; D000098662:Vasodilator-Stimulated Phosphoprotein; C070571:ML 7; C071058:moesin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium