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

Li HH, Li J, Wasserloos KJ, Wallace C, Sullivan MG, Bauer PM, Stolz DB, Lee JS, Watkins SC, St Croix CM, Pitt BR, Zhang LM - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

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Inhibition of macropinocytosis prevents Alexa488-BSA uptake by rat pulmonary endothelial cells.(A) RPMEC were serum starved for 4 h followed by pretreatment with various inhibitors of macropinocytosis for 30 min including amiloride (Na/H exchange inhibitor, 3 mM), Cytochalasin D (actin polymerization inhibitor, 10 µM), Gö6976 (PKC inhibitor, 10 µM), then cells were incubated with Alexa488-BSA (50 µg/ml) for 15 min and fixed with 2% paraformaldehyde for imaging. Images were taken by confocal microscopy (40× magnification). Representative images from three independent experiments are shown. (B) The fluorescence intensity was determined by Imaris software with data are present as mean ± SEM from three independent experiments. Significant differences from control (** P < 0.01, *** P < 0.001) were determined by one-way ANOVA followed by Tukey's multiple comparisons.
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pone-0081903-g006: Inhibition of macropinocytosis prevents Alexa488-BSA uptake by rat pulmonary endothelial cells.(A) RPMEC were serum starved for 4 h followed by pretreatment with various inhibitors of macropinocytosis for 30 min including amiloride (Na/H exchange inhibitor, 3 mM), Cytochalasin D (actin polymerization inhibitor, 10 µM), Gö6976 (PKC inhibitor, 10 µM), then cells were incubated with Alexa488-BSA (50 µg/ml) for 15 min and fixed with 2% paraformaldehyde for imaging. Images were taken by confocal microscopy (40× magnification). Representative images from three independent experiments are shown. (B) The fluorescence intensity was determined by Imaris software with data are present as mean ± SEM from three independent experiments. Significant differences from control (** P < 0.01, *** P < 0.001) were determined by one-way ANOVA followed by Tukey's multiple comparisons.

Mentions: The dispensability of cav-1 for at least a portion of albumin uptake was reminiscent of our recent experience with another ligand, Duffy antigen receptor for chemokine, and its role in chemokine internalization and transport across endothelium, previously assumed to involve a process of caveolar-dependent transcytosis [26]. Accordingly, we tested the drug sensitivity of a potential macropinocytic pathway and noted that uptake of Alexa488-BSA in RPMEC was significantly decreased by amiloride (inhibitor of Na/H exchange) and Gö6976, a protein kinase C inhibitor. In addition, cytochalasin D, an actin polymerization inhibitor also reduced uptake (Figure 6). We confirmed that albumin uptake was amiloride sensitive in both cav-1+/+ and cav-1-/- MLEC since 70% of albumin uptake was reduced in cav-1-/- cells while only 50% reduced in cav-1+/+ cells (data not shown). Collectively these data are consistent with macropinocytosis-like process accounting for a portion of albumin uptake in both rat and mouse endothelium.


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

Li HH, Li J, Wasserloos KJ, Wallace C, Sullivan MG, Bauer PM, Stolz DB, Lee JS, Watkins SC, St Croix CM, Pitt BR, Zhang LM - PLoS ONE (2013)

Inhibition of macropinocytosis prevents Alexa488-BSA uptake by rat pulmonary endothelial cells.(A) RPMEC were serum starved for 4 h followed by pretreatment with various inhibitors of macropinocytosis for 30 min including amiloride (Na/H exchange inhibitor, 3 mM), Cytochalasin D (actin polymerization inhibitor, 10 µM), Gö6976 (PKC inhibitor, 10 µM), then cells were incubated with Alexa488-BSA (50 µg/ml) for 15 min and fixed with 2% paraformaldehyde for imaging. Images were taken by confocal microscopy (40× magnification). Representative images from three independent experiments are shown. (B) The fluorescence intensity was determined by Imaris software with data are present as mean ± SEM from three independent experiments. Significant differences from control (** P < 0.01, *** P < 0.001) were determined by one-way ANOVA followed by Tukey's multiple comparisons.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842245&req=5

pone-0081903-g006: Inhibition of macropinocytosis prevents Alexa488-BSA uptake by rat pulmonary endothelial cells.(A) RPMEC were serum starved for 4 h followed by pretreatment with various inhibitors of macropinocytosis for 30 min including amiloride (Na/H exchange inhibitor, 3 mM), Cytochalasin D (actin polymerization inhibitor, 10 µM), Gö6976 (PKC inhibitor, 10 µM), then cells were incubated with Alexa488-BSA (50 µg/ml) for 15 min and fixed with 2% paraformaldehyde for imaging. Images were taken by confocal microscopy (40× magnification). Representative images from three independent experiments are shown. (B) The fluorescence intensity was determined by Imaris software with data are present as mean ± SEM from three independent experiments. Significant differences from control (** P < 0.01, *** P < 0.001) were determined by one-way ANOVA followed by Tukey's multiple comparisons.
Mentions: The dispensability of cav-1 for at least a portion of albumin uptake was reminiscent of our recent experience with another ligand, Duffy antigen receptor for chemokine, and its role in chemokine internalization and transport across endothelium, previously assumed to involve a process of caveolar-dependent transcytosis [26]. Accordingly, we tested the drug sensitivity of a potential macropinocytic pathway and noted that uptake of Alexa488-BSA in RPMEC was significantly decreased by amiloride (inhibitor of Na/H exchange) and Gö6976, a protein kinase C inhibitor. In addition, cytochalasin D, an actin polymerization inhibitor also reduced uptake (Figure 6). We confirmed that albumin uptake was amiloride sensitive in both cav-1+/+ and cav-1-/- MLEC since 70% of albumin uptake was reduced in cav-1-/- cells while only 50% reduced in cav-1+/+ cells (data not shown). Collectively these data are consistent with macropinocytosis-like process accounting for a portion of albumin uptake in both rat and mouse endothelium.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

Show MeSH
Related in: MedlinePlus