title: Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.
abstract: Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.
authors: Institute of Physiology, Charité - Universitätsmedizin Berlin, Berlin, Germany; German Heart Institute Berlin, Berlin, Germany; The Keenan Research Centre for Biomedical Science of St. Michael's, Toronto, Ontario, Canada; Departments of Surgery and Physiology, University of Toronto, Toronto, Ontario, Canada;
authors: Wolfgang M
authors: WM
authors: Kuebler
authors: Institute of Physiology, Charité - Universitätsmedizin Berlin, Berlin, Germany;
authors: Claudia
authors: C
authors: Wittenberg
authors: The Keenan Research Centre for Biomedical Science of St. Michael's, Toronto, Ontario, Canada; Interdepartmental Division of Critical Care, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario, Canada;
authors: Warren L
authors: WL
authors: Lee
authors: Division of Pulmonary Pharmacology, Research Center Borstel, Borstel, Germany;
authors: Eike
authors: E
authors: Reppien
authors: The Keenan Research Centre for Biomedical Science of St. Michael's, Toronto, Ontario, Canada;
authors: Neil M
authors: NM
authors: Goldenberg
authors: Division of Pulmonary Pharmacology, Research Center Borstel, Borstel, Germany;
authors: Karsten
authors: K
authors: Lindner
authors: The Keenan Research Centre for Biomedical Science of St. Michael's, Toronto, Ontario, Canada;
authors: Yizhuo
authors: Y
authors: Gao
authors: Institute of Immunology, University of Kiel, Kiel, Germany;
authors: Supandi
authors: S
authors: Winoto-Morbach
authors: Institute of Immunology and Experimental Therapy, Wroclaw, Poland;
authors: Marek
authors: M
authors: Drab
authors: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DLZ), Hannover, Germany; Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy), Hannover, Germany; and.
authors: Christian
authors: C
authors: Mühlfeld
authors: Division of Pulmonary Pharmacology, Research Center Borstel, Borstel, Germany;
authors: Heike
authors: H
authors: Dombrowsky
authors: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DLZ), Hannover, Germany; Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy), Hannover, Germany; and.
authors: Matthias
authors: M
authors: Ochs
authors: Institute of Immunology, University of Kiel, Kiel, Germany;
authors: Stefan
authors: S
authors: Schütze
authors: Division of Pulmonary Pharmacology, Research Center Borstel, Borstel, Germany; Institute of Pharmacology and Toxicology, RWTH Aachen, Aachen, Germany suhlig@ukaachen.de.
authors: Stefan
authors: S
authors: Uhlig
mesh_terms: D000418:Albumins / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D006801:Humans; D008168:Lung / Q000098:blood supply; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism; D055806:Microvessels / Q000166:cytology; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013917:Thrombin / Q000502:physiology; D057900:Transcytosis
chemical_list: D000418:Albumins; D051242:Caveolin 1; D013108:Sphingomyelin Phosphodiesterase; D013917:Thrombin
keywords: acid sphingomyelinase; acute lung injury; caveolin-1; ceramide; transcytosis