Co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and Rac.
Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.
Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
Susan M
SM
Armstrong
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Vahid
V
Khajoee
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Changsen
C
Wang
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Tieling
T
Wang
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Jayesh
J
Tigdi
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Jun
J
Yin
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Wolfgang M
WM
Kuebler
Departments of Microbiology and Infectious Diseases and Medicine, University of Calgary, Calgary, Alberta, Canada.
Mark
M
Gillrie
Departments of Microbiology and Infectious Diseases and Medicine, University of Calgary, Calgary, Alberta, Canada.
Shevaun P
SP
Davis
Departments of Microbiology and Infectious Diseases and Medicine, University of Calgary, Calgary, Alberta, Canada.
May
M
Ho
Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; Division of Respirology and Interdepartmental Division of Critical Care Medicine, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. Electronic address: warren.lee@utoronto.ca.
Warren L
WL
Lee
D008841:Actin Cytoskeleton / Q000502:physiology; D000418:Albumins / Q000493:pharmacokinetics*; D000818:Animals; D002199:Capillary Permeability / Q000502:physiology*; D051242:Caveolin 1 / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors*; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D019276:Glycocalyx / Q000378:metabolism; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D055806:Microvessels; D050600:SNARE Proteins / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D057900:Transcytosis / Q000187:drug effects / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000418:Albumins; D051242:Caveolin 1; D017630:Connexins; D006835:Hydrazones; D008246:Lysophospholipids; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D050600:SNARE Proteins; C060506:sphingosine 1-phosphate; D020744:rac GTP-Binding Proteins; D034281:Dynamins; D013110:Sphingosine