Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells

• Published in: PloS one Vol. 8; no. 11; p. e81903
• Main Authors: Li, Hui-Hua, Li, Jin, Wasserloos, Karla J, Wallace, Callen, Sullivan, Mara G, Bauer, Philip M, Stolz, Donna B, Lee, Janet S, Watkins, Simon C, St Croix, Claudette M, Pitt, Bruce R, Zhang, Li-Ming
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27.11.2013; Public Library of Science (PLoS)
• Subjects: Actin; Albumin; Albumins - metabolism; Amiloride; Anesthesiology; Animals; Base Sequence; Biology; Caveolae; Caveolae - physiology; Caveolin; Caveolin 1 - genetics; Caveolin 1 - metabolism; Caveolin-1; Cells, Cultured; Chemokines; Cholera; Clathrin; Coated pits; Confocal; Cytochalasin D; DNA Primers; Electron microscopy; Endocytosis; Endothelial cells; Endothelium; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Enzyme inhibitors; Fluorescence; Fluorescence microscopy; Gold; Infections; Inhibitors; Internalization; Laboratory animals; Localization; Lung - blood supply; Lungs; Medicine; Mice; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Microvasculature; Occupational health; Pathways; Penicillin; Permeability; Pharmacology; Phosphorylation; Pinocytosis; Pits; Polymerization; Protein kinase C; Protein transport; Proteins; Public health; Pulmonary arteries; Pulmonary artery; Rats; Reflectance; RNA, Small Interfering - genetics; Rodents; siRNA; Sodium; Water-borne diseases; Waterborne diseases; United States > US; Pittsburgh Pennsylvania; Pennsylvania
• DOI: 10.1371/journal.pone.0081903
• ISSN: 1932-6203

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1(-/-) mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1(-/-) mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1(-/-) MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.