Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement.
Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Irina A
IA
Kolosova
Tamara
T
Mirzapoiazova
Djanybek
D
Adyshev
Peter
P
Usatyuk
Lewis H
LH
Romer
Jeffrey R
JR
Jacobson
Viswanathan
V
Natarajan
David B
DB
Pearse
Joe G N
JG
Garcia
Alexander D
AD
Verin
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000502:physiology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000502:physiology; D006801:Humans; D007365:Intercellular Junctions / Q000187:drug effects; D008840:Microfilament Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D015398:Signal Transduction; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D043263:Myosin-Light-Chain Phosphatase; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D002118:Calcium