Role of myosin light-chain phosphorylation in endothelial cell retraction.
Endothelial cells retract centripetally when they are exposed to histamine and when extracellular calcium is chelated. This centripetal retraction implies that a centripetal tension must be expressed in the cells. We asked whether phosphorylation of the light chain of myosin (MLC) was important for the retraction to occur, and, by inference, expression of the tension. In human umbilical vein endothelial (HUVE) cells and in porcine pulmonary artery endothelial (PPAE) cells tryptic peptide maps indicated that MLC was phosphorylated by myosin light-chain kinase (MLCK). Activity of MLCK is inhibited by ML-9, a kinase inhibitor with relative specificity for MLCK, and when MLCK is phosphorylated by the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent kinase. Pretreatment of HUVE cells or PPAE cells with ML-9 or forskolin-aminophylline (to increase cell cAMP) reduced basal MLC phosphorylation and prevented an expected increase in MLC phosphorylation following exposure of HUVE cells to histamine. Pretreatment of HUVE cells with ML-9 or forskolin-aminophylline prevented HUVE cell retraction (measured as an increase in permeability of a monolayer of HUVE cells) in response to histamine. Pretreatment of PPAE cells with ML-9 or forskolin-aminophylline prevented PPAE cell retraction in response to chelation of extracellular calcium. These data support the hypothesis that phosphorylation of MLC is an important component of endothelial cell retraction.
Sheldon
R
R
Department of Medicine, University of Iowa College of Medicine, Iowa City 52242.
Moy
A
A
Lindsley
K
K
Shasby
S
S
Shasby
D M
DM
D000628:Aminophylline / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009218:Myosins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D011651:Pulmonary Artery; D013552:Swine; D014471:Umbilical Veins
D001381:Azepines; D010446:Peptide Fragments; C056218:ML 9; D005576:Colforsin; D000628:Aminophylline; D004533:Egtazic Acid; D006632:Histamine; D000242:Cyclic AMP; D009219:Myosin-Light-Chain Kinase; D009218:Myosins