title: Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells.
abstract: 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.
authors: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America ; Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors: Hui-Hua
authors: HH
authors: Li
authors: Jin
authors: J
authors: Li
authors: Karla J
authors: KJ
authors: Wasserloos
authors: Callen
authors: C
authors: Wallace
authors: Mara G
authors: MG
authors: Sullivan
authors: Philip M
authors: PM
authors: Bauer
authors: Donna B
authors: DB
authors: Stolz
authors: Janet S
authors: JS
authors: Lee
authors: Simon C
authors: SC
authors: Watkins
authors: Claudette M
authors: CM
authors: St Croix
authors: Bruce R
authors: BR
authors: Pitt
authors: Li-Ming
authors: LM
authors: Zhang
mesh_terms: D000418:Albumins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D021941:Caveolae / Q000502:physiology*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D008168:Lung / Q000098:blood supply*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010873:Pinocytosis; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats
chemical_list: D000418:Albumins; C495537:Cav1 protein, rat; D051242:Caveolin 1; D017931:DNA Primers; D034741:RNA, Small Interfering