Mechanisms of sodium fluoride-induced endothelial cell barrier dysfunction: role of MLC phosphorylation.
NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicating endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of Ca(2+)/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction.
Wang
P
P
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224-6801, USA.
Verin
A D
AD
Birukova
A
A
Gilbert-McClain
L I
LI
Jacobs
K
K
Garcia
J G
JG
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D003599:Cytoskeleton / Q000378:metabolism; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology*; D012969:Sodium Fluoride / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D012969:Sodium Fluoride; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D020741:rho GTP-Binding Proteins; D002118:Calcium