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

Li, HH and Li, J and Wasserloos, KJ and Wallace, C and Sullivan, MG and Bauer, PM and Stolz, DB and Lee, JS and Watkins, SC and St Croix, CM and Pitt, BR and Zhang, LM (2013) Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells. PLoS ONE, 8 (11).

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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. © 2013 Li et al.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Li, HHhul18@pitt.eduHUL18
Li, J
Wasserloos, KJ
Wallace, Cctw14@pitt.eduCTW14
Sullivan, MG
Bauer, PM
Stolz, DBdonna.stolz@pitt.eduDSTOLZ
Lee, JSjsl26@pitt.eduJSL26
Watkins, SCsimon.watkins@pitt.eduSWATKINS
St Croix, CMclaudette.stcroix@pitt.eduCLS13
Pitt, BRbrucep@pitt.eduBRUCEP
Zhang, LMliz19@pitt.eduLIZ19
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorZhao, You-YangUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, or Units > Center for Biologic Imaging
Date: 27 November 2013
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 8
Number: 11
DOI or Unique Handle: 10.1371/journal.pone.0081903
Schools and Programs: Graduate School of Public Health > Environmental and Occupational Health
School of Medicine > Anesthesiology
School of Medicine > Cell Biology
School of Medicine > Critical Care Medicine
School of Medicine > Surgery
Refereed: Yes
Date Deposited: 30 Jan 2014 18:10
Last Modified: 02 Feb 2019 16:58
URI: http://d-scholarship.pitt.edu/id/eprint/20416

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