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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 phosphorylation of eNOS-Thr497 and decreases NO production in BAEC. BAEC were treated with (A) 30 μM sodium arsenite (NaAsO2) for shorter times (4, 8, or 24 h), (B) for longer times (up to 36 h), or (C) with various doses of sodium arsenite (10, 20, or 30 μM) for 4 h (C). Control cells (0 h or 0 μM) were treated with vehicle (H2O) only. The cells were lysed, and the amounts of phosphorylated eNOS (p-eNOS) at multiple sites were measured by Western blot analysis using antibodies specific for eNOS phosphorylated at Ser116 (p-eNOS-Ser116), p-eNOS-Thr497, or p-eNOS-Ser1179. The blots shown are representative of at least three experiments. Densitometry was used to quantify p-eNOS-Ser116, p-eNOS-Thr497, or p-eNOS-Ser1179 relative to the total protein bands, and the graphs show the mean fold alterations above or below control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (D) After BAEC were treated with 30 μM sodium arsenite for the indicated times, NO released by the cells was measured by the Griess method. Each bar represents the mean NO production (after normalization to total cellular protein) as fold decreases below control (vehicle) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.
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f1-bt-22-510: Arsenite increases phosphorylation of eNOS-Thr497 and decreases NO production in BAEC. BAEC were treated with (A) 30 μM sodium arsenite (NaAsO2) for shorter times (4, 8, or 24 h), (B) for longer times (up to 36 h), or (C) with various doses of sodium arsenite (10, 20, or 30 μM) for 4 h (C). Control cells (0 h or 0 μM) were treated with vehicle (H2O) only. The cells were lysed, and the amounts of phosphorylated eNOS (p-eNOS) at multiple sites were measured by Western blot analysis using antibodies specific for eNOS phosphorylated at Ser116 (p-eNOS-Ser116), p-eNOS-Thr497, or p-eNOS-Ser1179. The blots shown are representative of at least three experiments. Densitometry was used to quantify p-eNOS-Ser116, p-eNOS-Thr497, or p-eNOS-Ser1179 relative to the total protein bands, and the graphs show the mean fold alterations above or below control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (D) After BAEC were treated with 30 μM sodium arsenite for the indicated times, NO released by the cells was measured by the Griess method. Each bar represents the mean NO production (after normalization to total cellular protein) as fold decreases below control (vehicle) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.

Mentions: Because chronic (>24 h) exposure of arsenite has shown the increased vascular dysfunction mainly by decreasing eNOS-Ser1179 phosphorylation-mediated NO production, we asked whether acute exposure of arsenite also decreases NO production via decreased eNOS-Ser1179 phosphorylation. As shown in Fig. 1A, however, arsenite (30 μM) acutely (as early as 4 h) increased eNOS-Thr497 phosphorylation, but not of eNOS-Ser1179 phosphorylation, although its longer (at 24 h) exposure considerably decreased eNOS-Ser1179 phosphorylation. No alterations in eNOS-Ser116 phosphorylation and eNOS expression were found under these conditions. However, longer (36 h) exposure of arsenite significantly decreased eNOS expression (Fig. 1B), which was accompanied by significantly decreased cell viability (data not shown). We also found that arsenite acutely increased eNOS-Thr497 phosphorylation in a dose-dependent manner (Fig. 1C). Consistent with phosphorylation status of eNOS-Thr497, arsenite decreased NO production in a time-dependent manner (Fig. 1D). Because a significant increase in eNOS-Thr497 phosphorylation was found in BAEC acutely exposed to arsenite, all subsequent experiments were accomplished with 30 μM arsenite treatment for 4 h, unless otherwise specifically stated.


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 phosphorylation of eNOS-Thr497 and decreases NO production in BAEC. BAEC were treated with (A) 30 μM sodium arsenite (NaAsO2) for shorter times (4, 8, or 24 h), (B) for longer times (up to 36 h), or (C) with various doses of sodium arsenite (10, 20, or 30 μM) for 4 h (C). Control cells (0 h or 0 μM) were treated with vehicle (H2O) only. The cells were lysed, and the amounts of phosphorylated eNOS (p-eNOS) at multiple sites were measured by Western blot analysis using antibodies specific for eNOS phosphorylated at Ser116 (p-eNOS-Ser116), p-eNOS-Thr497, or p-eNOS-Ser1179. The blots shown are representative of at least three experiments. Densitometry was used to quantify p-eNOS-Ser116, p-eNOS-Thr497, or p-eNOS-Ser1179 relative to the total protein bands, and the graphs show the mean fold alterations above or below control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (D) After BAEC were treated with 30 μM sodium arsenite for the indicated times, NO released by the cells was measured by the Griess method. Each bar represents the mean NO production (after normalization to total cellular protein) as fold decreases below control (vehicle) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-bt-22-510: Arsenite increases phosphorylation of eNOS-Thr497 and decreases NO production in BAEC. BAEC were treated with (A) 30 μM sodium arsenite (NaAsO2) for shorter times (4, 8, or 24 h), (B) for longer times (up to 36 h), or (C) with various doses of sodium arsenite (10, 20, or 30 μM) for 4 h (C). Control cells (0 h or 0 μM) were treated with vehicle (H2O) only. The cells were lysed, and the amounts of phosphorylated eNOS (p-eNOS) at multiple sites were measured by Western blot analysis using antibodies specific for eNOS phosphorylated at Ser116 (p-eNOS-Ser116), p-eNOS-Thr497, or p-eNOS-Ser1179. The blots shown are representative of at least three experiments. Densitometry was used to quantify p-eNOS-Ser116, p-eNOS-Thr497, or p-eNOS-Ser1179 relative to the total protein bands, and the graphs show the mean fold alterations above or below control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. (D) After BAEC were treated with 30 μM sodium arsenite for the indicated times, NO released by the cells was measured by the Griess method. Each bar represents the mean NO production (after normalization to total cellular protein) as fold decreases below control (vehicle) (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01.
Mentions: Because chronic (>24 h) exposure of arsenite has shown the increased vascular dysfunction mainly by decreasing eNOS-Ser1179 phosphorylation-mediated NO production, we asked whether acute exposure of arsenite also decreases NO production via decreased eNOS-Ser1179 phosphorylation. As shown in Fig. 1A, however, arsenite (30 μM) acutely (as early as 4 h) increased eNOS-Thr497 phosphorylation, but not of eNOS-Ser1179 phosphorylation, although its longer (at 24 h) exposure considerably decreased eNOS-Ser1179 phosphorylation. No alterations in eNOS-Ser116 phosphorylation and eNOS expression were found under these conditions. However, longer (36 h) exposure of arsenite significantly decreased eNOS expression (Fig. 1B), which was accompanied by significantly decreased cell viability (data not shown). We also found that arsenite acutely increased eNOS-Thr497 phosphorylation in a dose-dependent manner (Fig. 1C). Consistent with phosphorylation status of eNOS-Thr497, arsenite decreased NO production in a time-dependent manner (Fig. 1D). Because a significant increase in eNOS-Thr497 phosphorylation was found in BAEC acutely exposed to arsenite, all subsequent experiments were accomplished with 30 μM arsenite treatment for 4 h, unless otherwise specifically stated.

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