Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase.
Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125(FAK)), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.
Mucha
David R
DR
The Benjamin W. Zweifach Microcirculation Laboratories, Veterans Affairs Medical Center, Tucson, Arizona 85723, USA.
Myers
Carter L
CL
Schaeffer
Richard C
RC
D000818:Animals; D016227:Benzoquinones; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases; D047029:Lactams, Macrocyclic; D008387:Marine Toxins; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010080:Oxazoles / Q000494:pharmacology; D051419:Paxillin; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D019061:src-Family Kinases / Q000378:metabolism*
D016227:Benzoquinones; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D008387:Marine Toxins; D018994:Myosin Light Chains; D010080:Oxazoles; D051419:Paxillin; D010750:Phosphoproteins; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; C059041:calyculin A; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase