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.
Highlights
Caveolae are flask-shaped membrane invaginations of the plasma membrane and caveolin-1 acts as the major structural protein and biochemical marker of caveolae [1,2,3]
From original gold-labeled albumin electron microscopy studies in intact wild type animals [17,18] to more recent investigations with similar technology in cav-1 null mice [19,20] as well as efforts in isolated cultured lung endothelial cells [21,22], a concensus has emerged regarding the importance of caveolaedependent transcytosis of albumin in the pulmonary endothelium
Mouse lung endothelial cells (MLEC) were isolated from wild type or caveolin-1 null mice [28] by immunobeads coated with anti-mouse PECAM (CD31) antibody (BD Pharmingen, San Diego, CA, USA) followed by cell sorting with fluorescently-labeled acetylated-Low Density Lipoprotein (Dil-AcLDL, Biomedical Technologies Inc., Stoughton, MA, USA) uptake
Summary
Caveolae are flask-shaped membrane invaginations of the plasma membrane and caveolin-1 (cav-1, a 22-kDa protein) acts as the major structural protein and biochemical marker of caveolae [1,2,3]. In cav-1 null mice that are devoid of caveolae, occasional invaginations with electron dense diaphragms in large vessels of the lung were observed [19] This could account for the minor fraction of gold-labeled albumin that was taken up in fluid phase by endocytosis [18] or the preliminary observations of caveolae-independent goldlabeled albumin uptake in the lung by Predescu et al [23]. The original observation of normal albumin transport in the choroid plexus (ascribed to transcytosis) in cav-1 null mice [19] suggests possible caveolae-independent transport of albumin This latter observation is distinctly different from the hyperpermeability of albumin in cav-1 null mice [24,25] that is sensitive to nitric oxide synthase inhibitors and in which the role of enhanced NO biosynthesis on paracellular pathways has been elucidated in lung endothelium [21]
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