Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability.
The actomyosin complex is the major cytoskeletal component that controls cell contraction. In this study, we investigated the effects of actomyosin interaction on endothelial barrier function and gap formation. Activated myosin light chain kinase (MLCK) protein was transferred into coronary venular endothelial cell (CVEC) monolayers. Uptake of the activated protein resulted in a significant shift in myosin light chain (MLC) from an unphosphorylated to a diphosphorylated form. In addition, MLCK induced a hyperpermeability response of the monolayer as measured by albumin transendothelial flux. Microscopic examination of MLCK-treated CVECs revealed widespread gap formation in the monolayer, loss of peripheral beta-catenin, and increases in actin stress fibers. Inhibition of all of the above responses by a specific MLCK inhibitor suggests they are the direct result of exogenously added MLCK. These data suggest that activation of MLCK in CVECs causes phosphorylation of MLC and contraction of CVECs, resulting in gap formation and concomitant increases in permeability. This study uses a novel technique to measure the effects of an activated kinase on both its substrate and cellular morphology and function through direct transference into endothelial cells.
Tinsley
J H
JH
Departments of Surgery and Medical Physiology, Texas A&M University System Health Science Center, Temple, Texas 76504, USA. jht@tamu.edu
De Lanerolle
P
P
Wilson
E
E
Ma
W
W
Yuan
S Y
SY
D000199:Actins / Q000378:metabolism; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002645:Chickens; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008387:Marine Toxins; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010080:Oxazoles / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011073:Polyamines / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism; D015534:Trans-Activators; D015854:Up-Regulation; D051176:beta Catenin
D000199:Actins; D001381:Azepines; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D018994:Myosin Light Chains; D010080:Oxazoles; D011073:Polyamines; D012709:Serum Albumin; D015534:Trans-Activators; D051176:beta Catenin; C056218:ML 9; C059041:calyculin A; D009219:Myosin-Light-Chain Kinase