title: The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?
abstract: Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.
authors.affiliation: Vascular Biology Center, Georgia Health Sciences University, Augusta, Georgia 30912, USA. nbogatcheva@georgiahealth.edu
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Bogatcheva
authors.forename: Marina A
authors.initials: MA
authors.lastname: Zemskova
authors.forename: Christophe
authors.initials: C
authors.lastname: Poirier
authors.forename: Tamara
authors.initials: T
authors.lastname: Mirzapoiazova
authors.forename: Irina
authors.initials: I
authors.lastname: Kolosova
authors.forename: Anne R
authors.initials: AR
authors.lastname: Bresnick
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
mesh_terms: D000577:Amides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003911:Dextrans / Q000378:metabolism; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004731:Endotoxins / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000098:blood supply*; D055806:Microvessels / Q000187:drug effects* / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000577:Amides; D003911:Dextrans; D004731:Endotoxins; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C015219:fluorescein isothiocyanate dextran; C108830:Y 27632; D005576:Colforsin; C050086:N(6)-benzoyl-cyclic AMP; C044101:endotoxin, Escherichia coli; D000242:Cyclic AMP; C515851:ROCK1 protein, human; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium