Phorbol esters increase MLC phosphorylation and actin remodeling in bovine lung endothelium without increased contraction.
Direct protein kinase C (PKC) activation with phorbol myristate acetate (PMA) results in the loss of endothelial monolayer integrity in bovine lung endothelial cells (EC) but produces barrier enhancement in human lung endothelium. To extend these findings, we studied EC contractile events and observed a 40% increase in myosin light chain (MLC) phosphorylation in bovine endothelium following PMA challenge. The increase in PMA-mediated MLC phosphorylation occurred at sites distinct from Ser19/Thr18, sites catalyzed by MLC kinase (MLCK), and immunoblotting with antibodies specific to phosphorylated Ser19/Thr18 demonstrated profound time-dependent Ser19/Thr18 dephosphorylation. These events occurred in conjunction with rearrangement of stress fibers into a grid-like network, but without an increase in cellular contraction as measured by silicone membrane wrinkling assay. The PMA-induced MLC dephosphorylation was not due to kinase inhibition but, rather, correlated with rapid increases in myosin-associated phosphatase 1 (PPase 1) activity. These data suggest that PMA-mediated EC barrier regulation may involve dual mechanisms that alter MLC phosphorylation. The increase in bovine MLC phosphorylation likely occurs via direct PKC-dependent MLC phosphorylation in conjunction with decreases in Ser19/Thr18 phosphorylation catalyzed by MLCK due to PMA-induced increases in PPase 1 activity. Together, these events result in stress fiber destabilization and profound actin rearrangement in bovine endothelium, which may result in the physiological alterations observed in these models.
Bogatcheva
Natalia V
NV
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, MD 21218, USA.
Verin
Alexander D
AD
Wang
Peiyi
P
Birukova
Anna A
AA
Birukov
Konstantin G
KG
Mirzopoyazova
Tamara
T
Adyshev
Djanybek M
DM
Chiang
Eddie T
ET
Crow
Michael T
MT
Garcia
Joe G N
JG
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D008168:Lung / Q000187:drug effects / Q000502:physiology*; D016296:Mutagenesis; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011652:Pulmonary Circulation / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014982:Xenopus laevis
D000199:Actins; D017931:DNA Primers; D018994:Myosin Light Chains; D012333:RNA, Messenger; D013755:Tetradecanoylphorbol Acetate