Limits...
Arsenite Acutely Decreases Nitric Oxide Production via the ROS-Protein Phosphatase 1-Endothelial Nitric Oxide Synthase-Thr(497) Signaling Cascade.

Seo J, Lee JY, Sung MS, Byun CJ, Cho DH, Lee HJ, Park JH, Cho HS, Cho SJ, Jo I - Biomol Ther (Seoul) (2014)

Bottom Line: Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr(497) phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr(497) phosphorylation.In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr(497) phosphorylation.Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710 ; Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan 570-749.

ABSTRACT
Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser(1179) in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr(497), but not of eNOS-Ser(116) or eNOS-Ser(1179), which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr(497) phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr(497) phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

No MeSH data available.


Related in: MedlinePlus

Arsenite increases ROS production and antioxidant NAC reverses arsenite-induced increased eNOS-Thr497 phosphorylation. (A) BAEC were treated with 30 μM sodium arsenite for the indicated times (0, 0.5, 1, 2, or 4 h). ROS was measured by DCFH-DA method using FACSCalibur flow cytometer. Each bar represents mean ROS production as fold increases above control (0 h) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (B) Cells were also preincubated with vehicle or 10 mM NAC for 0.5 h, and then further treated with 30 μM sodium arsenite for 4 h. The level of p-eNOS-Thr497 was measured by Western blot analysis as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold increase above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256030&req=5

f2-bt-22-510: Arsenite increases ROS production and antioxidant NAC reverses arsenite-induced increased eNOS-Thr497 phosphorylation. (A) BAEC were treated with 30 μM sodium arsenite for the indicated times (0, 0.5, 1, 2, or 4 h). ROS was measured by DCFH-DA method using FACSCalibur flow cytometer. Each bar represents mean ROS production as fold increases above control (0 h) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (B) Cells were also preincubated with vehicle or 10 mM NAC for 0.5 h, and then further treated with 30 μM sodium arsenite for 4 h. The level of p-eNOS-Thr497 was measured by Western blot analysis as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold increase above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.

Mentions: Several studies have demonstrated that arsenite generates ROS that leads to alteration in the activity of proteins; for example, arsenite increases ROS, resulting in increased expression or secretion of vascular endothelial growth factor (VEGF) in cancer cells and brain microvascular pericytes, respectively (Gao et al., 2004; Park et al., 2009). Therefore, we first evaluated whether arsenite induced ROS production in BAEC. As shown in Fig 2A, arsenite acutely (as early as 0.5 h) increased ROS production by ∼1.75 fold and this increase was maintained up to 4 h. Next, we tested whether increased ROS affected eNOS-Thr497 phosphorylation. Pretreatment with antioxidant NAC (10 mM) for 0.5 h almost completely reversed the arsenite-induced increase in eNOS-Thr497 phosphorylation (Fig. 2B), suggesting that ROS mediates the observed effect by arsenite.


Arsenite Acutely Decreases Nitric Oxide Production via the ROS-Protein Phosphatase 1-Endothelial Nitric Oxide Synthase-Thr(497) Signaling Cascade.

Seo J, Lee JY, Sung MS, Byun CJ, Cho DH, Lee HJ, Park JH, Cho HS, Cho SJ, Jo I - Biomol Ther (Seoul) (2014)

Arsenite increases ROS production and antioxidant NAC reverses arsenite-induced increased eNOS-Thr497 phosphorylation. (A) BAEC were treated with 30 μM sodium arsenite for the indicated times (0, 0.5, 1, 2, or 4 h). ROS was measured by DCFH-DA method using FACSCalibur flow cytometer. Each bar represents mean ROS production as fold increases above control (0 h) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (B) Cells were also preincubated with vehicle or 10 mM NAC for 0.5 h, and then further treated with 30 μM sodium arsenite for 4 h. The level of p-eNOS-Thr497 was measured by Western blot analysis as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold increase above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256030&req=5

f2-bt-22-510: Arsenite increases ROS production and antioxidant NAC reverses arsenite-induced increased eNOS-Thr497 phosphorylation. (A) BAEC were treated with 30 μM sodium arsenite for the indicated times (0, 0.5, 1, 2, or 4 h). ROS was measured by DCFH-DA method using FACSCalibur flow cytometer. Each bar represents mean ROS production as fold increases above control (0 h) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (B) Cells were also preincubated with vehicle or 10 mM NAC for 0.5 h, and then further treated with 30 μM sodium arsenite for 4 h. The level of p-eNOS-Thr497 was measured by Western blot analysis as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold increase above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.
Mentions: Several studies have demonstrated that arsenite generates ROS that leads to alteration in the activity of proteins; for example, arsenite increases ROS, resulting in increased expression or secretion of vascular endothelial growth factor (VEGF) in cancer cells and brain microvascular pericytes, respectively (Gao et al., 2004; Park et al., 2009). Therefore, we first evaluated whether arsenite induced ROS production in BAEC. As shown in Fig 2A, arsenite acutely (as early as 0.5 h) increased ROS production by ∼1.75 fold and this increase was maintained up to 4 h. Next, we tested whether increased ROS affected eNOS-Thr497 phosphorylation. Pretreatment with antioxidant NAC (10 mM) for 0.5 h almost completely reversed the arsenite-induced increase in eNOS-Thr497 phosphorylation (Fig. 2B), suggesting that ROS mediates the observed effect by arsenite.

Bottom Line: Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr(497) phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr(497) phosphorylation.In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr(497) phosphorylation.Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, School of Medicine, Ewha Womans University, Seoul 158-710 ; Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan 570-749.

ABSTRACT
Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser(1179) in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr(497), but not of eNOS-Ser(116) or eNOS-Ser(1179), which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr(497) phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr(497) phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

No MeSH data available.


Related in: MedlinePlus