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Regulation of β-adrenergic receptor trafficking and lung microvascular endothelial cell permeability by Rab5 GTPase.

Yang J, Sun H, Zhang J, Hu M, Wang J, Wu G, Wang G - Int. J. Biol. Sci. (2015)

Bottom Line: Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs.Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability.All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

ABSTRACT
Rab5 GTPase modulates the trafficking of the cell surface receptors, including G protein-coupled β-adrenergic receptors (β-ARs). Here, we have determined the role of Rab5 in regulating the internalization of β-ARs in lung microvascular endothelial cells (LMECs) and in maintaining the integrity and permeability of endothelial cell barrier. Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs. Furthermore, the activation of β-ARs, particularly β2-AR, is able to protect the LMEC permeability from LPS injury. Moreover, siRNA-mediated knockdown of Rab5 inhibits both the basal and agonist-provoked internalization of β-ARs, therefore, enhancing the cell surface expression of the receptors and receptor-mediated ERK1/2 activation. Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability. All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

No MeSH data available.


Related in: MedlinePlus

Effects of LPS on the cell permeability and the cell surface expression of β-ARs in LMECs. (A) LPS treatment decreased the CI of LMECs in a dose- and time-dependent manner. The cells were cultured and the CI was measured in the iCELLigence System as described in “Materials and methods”. The images shown are representative CI traces after treatment with LPS at concentrations of 0 (black), 5 μg/ml (blue), 10 μg/ml (red), 20 μg/ml (green) and 50 μg/ml (pink), and were taken from one of four independent measurements. (B) Quantification of total cell surface β-ARs after LPS treatment. The mean values of specific [3H]-CGP12177 binding were 1859 ± 221, 1605 ± 176, 1467 ± 158, 1015 ± 146, and 997 ± 128 cpm at 0, 2, 4, 6, and 8 h groups, respectively. The data are expressed as the means ± S. E. (n=4). *P< 0.05 versus the 0 h group and ^P< 0.05 versus the 4 h group.
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Figure 1: Effects of LPS on the cell permeability and the cell surface expression of β-ARs in LMECs. (A) LPS treatment decreased the CI of LMECs in a dose- and time-dependent manner. The cells were cultured and the CI was measured in the iCELLigence System as described in “Materials and methods”. The images shown are representative CI traces after treatment with LPS at concentrations of 0 (black), 5 μg/ml (blue), 10 μg/ml (red), 20 μg/ml (green) and 50 μg/ml (pink), and were taken from one of four independent measurements. (B) Quantification of total cell surface β-ARs after LPS treatment. The mean values of specific [3H]-CGP12177 binding were 1859 ± 221, 1605 ± 176, 1467 ± 158, 1015 ± 146, and 997 ± 128 cpm at 0, 2, 4, 6, and 8 h groups, respectively. The data are expressed as the means ± S. E. (n=4). *P< 0.05 versus the 0 h group and ^P< 0.05 versus the 4 h group.

Mentions: We then determined the effect of LPS treatment on the barrier permeability of LMECs by measuring the CI. Lung EC barrier dysfunction is characterized by increased permeation of fluid and macromolecules into interstitial and alveolar tissues, and is associated with a decreased CI 23. To determine whether LPS treatment affects the EC barrier function, we challenged confluent cell monolayers with different doses of LPS (0, 5, 10, 20, or 50 µg/ml) for 9 h. We found that the CI decreased between 0 h and 9 h in a concentration-dependent manner. At 5 µg/ml, LPS did not significantly affect the normalized CI after 4 h (CI = 0.9656; P>0.05 compared to baseline). However, by 6 and 8 h, 5 µg/ml LPS significantly reduced the CI (0.8945 and 0.8794, respectively; P<0.05 compared to baseline). At 10 µg/ml, LPS significantly decreased the normalized CI at 6 and 8 h (0.7567 and 0.7407, respectively; P<0.05 compared to either baseline) (Fig. 1A). Similar results were obtained when 20 µg/ml of LPS were used. The normalized CI markedly decreased when the cells were treated with 50 µg/ml LPS for 9 h as compared with control group (P<0.05, Fig. 1A). The normalized CI values in all LPS treatment groups were significantly decreased compared to the control group. These data demonstrated that the response to LPS treatment developed gradually and peaked after 6 h administration.


Regulation of β-adrenergic receptor trafficking and lung microvascular endothelial cell permeability by Rab5 GTPase.

Yang J, Sun H, Zhang J, Hu M, Wang J, Wu G, Wang G - Int. J. Biol. Sci. (2015)

Effects of LPS on the cell permeability and the cell surface expression of β-ARs in LMECs. (A) LPS treatment decreased the CI of LMECs in a dose- and time-dependent manner. The cells were cultured and the CI was measured in the iCELLigence System as described in “Materials and methods”. The images shown are representative CI traces after treatment with LPS at concentrations of 0 (black), 5 μg/ml (blue), 10 μg/ml (red), 20 μg/ml (green) and 50 μg/ml (pink), and were taken from one of four independent measurements. (B) Quantification of total cell surface β-ARs after LPS treatment. The mean values of specific [3H]-CGP12177 binding were 1859 ± 221, 1605 ± 176, 1467 ± 158, 1015 ± 146, and 997 ± 128 cpm at 0, 2, 4, 6, and 8 h groups, respectively. The data are expressed as the means ± S. E. (n=4). *P< 0.05 versus the 0 h group and ^P< 0.05 versus the 4 h group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495405&req=5

Figure 1: Effects of LPS on the cell permeability and the cell surface expression of β-ARs in LMECs. (A) LPS treatment decreased the CI of LMECs in a dose- and time-dependent manner. The cells were cultured and the CI was measured in the iCELLigence System as described in “Materials and methods”. The images shown are representative CI traces after treatment with LPS at concentrations of 0 (black), 5 μg/ml (blue), 10 μg/ml (red), 20 μg/ml (green) and 50 μg/ml (pink), and were taken from one of four independent measurements. (B) Quantification of total cell surface β-ARs after LPS treatment. The mean values of specific [3H]-CGP12177 binding were 1859 ± 221, 1605 ± 176, 1467 ± 158, 1015 ± 146, and 997 ± 128 cpm at 0, 2, 4, 6, and 8 h groups, respectively. The data are expressed as the means ± S. E. (n=4). *P< 0.05 versus the 0 h group and ^P< 0.05 versus the 4 h group.
Mentions: We then determined the effect of LPS treatment on the barrier permeability of LMECs by measuring the CI. Lung EC barrier dysfunction is characterized by increased permeation of fluid and macromolecules into interstitial and alveolar tissues, and is associated with a decreased CI 23. To determine whether LPS treatment affects the EC barrier function, we challenged confluent cell monolayers with different doses of LPS (0, 5, 10, 20, or 50 µg/ml) for 9 h. We found that the CI decreased between 0 h and 9 h in a concentration-dependent manner. At 5 µg/ml, LPS did not significantly affect the normalized CI after 4 h (CI = 0.9656; P>0.05 compared to baseline). However, by 6 and 8 h, 5 µg/ml LPS significantly reduced the CI (0.8945 and 0.8794, respectively; P<0.05 compared to baseline). At 10 µg/ml, LPS significantly decreased the normalized CI at 6 and 8 h (0.7567 and 0.7407, respectively; P<0.05 compared to either baseline) (Fig. 1A). Similar results were obtained when 20 µg/ml of LPS were used. The normalized CI markedly decreased when the cells were treated with 50 µg/ml LPS for 9 h as compared with control group (P<0.05, Fig. 1A). The normalized CI values in all LPS treatment groups were significantly decreased compared to the control group. These data demonstrated that the response to LPS treatment developed gradually and peaked after 6 h administration.

Bottom Line: Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs.Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability.All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

ABSTRACT
Rab5 GTPase modulates the trafficking of the cell surface receptors, including G protein-coupled β-adrenergic receptors (β-ARs). Here, we have determined the role of Rab5 in regulating the internalization of β-ARs in lung microvascular endothelial cells (LMECs) and in maintaining the integrity and permeability of endothelial cell barrier. Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs. Furthermore, the activation of β-ARs, particularly β2-AR, is able to protect the LMEC permeability from LPS injury. Moreover, siRNA-mediated knockdown of Rab5 inhibits both the basal and agonist-provoked internalization of β-ARs, therefore, enhancing the cell surface expression of the receptors and receptor-mediated ERK1/2 activation. Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability. All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

No MeSH data available.


Related in: MedlinePlus