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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 decreases PP1 activity and NAC reverses arsenite-induced decrease in PP1 activity. (A) BAEC were treated as described in the legends of Fig. 1. The level of PP1 was measured by Western blot analysis using anti-PP1α antibody as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold alteration above control (± S.D.). (B) BAEC were treated as described in the legend of Fig. 2. PP1 activity was measured by serine/threonine phosphatase assay kit (Promega), quantified by measuring the fluorescence intensity (at 485/530 nm) using FACSCalibur flow cytometer (BD), and normalized to the fluorescence intensity from the control experiment. The bar graph shows the mean fold decreases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05.
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f4-bt-22-510: Arsenite decreases PP1 activity and NAC reverses arsenite-induced decrease in PP1 activity. (A) BAEC were treated as described in the legends of Fig. 1. The level of PP1 was measured by Western blot analysis using anti-PP1α antibody as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold alteration above control (± S.D.). (B) BAEC were treated as described in the legend of Fig. 2. PP1 activity was measured by serine/threonine phosphatase assay kit (Promega), quantified by measuring the fluorescence intensity (at 485/530 nm) using FACSCalibur flow cytometer (BD), and normalized to the fluorescence intensity from the control experiment. The bar graph shows the mean fold decreases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05.

Mentions: To elucidate the molecular mechanism by which arsenite acutely increases PP1-mediated eNOS-Thr497 phosphorylation, we investigated whether arsenite indeed decreases the protein level and/or the activity of PP1 in BAEC. Although it was reported that arsenite treatment decreased the level of PP1α expression in human lymphoblastoid cells (Tapio et al., 2005), we failed to find decrease in the level of PP1α expression in arsenite-treated BAEC (Fig. 4A). Instead, arsenite significantly decreased PP1 activity by ∼15% (Fig. 4B). Furthermore, the arsenite-induced decrease in PP1 activity was almost completely recovered by the pretreatment of NAC, suggesting that ROS mediates decreased PP1 activity 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 decreases PP1 activity and NAC reverses arsenite-induced decrease in PP1 activity. (A) BAEC were treated as described in the legends of Fig. 1. The level of PP1 was measured by Western blot analysis using anti-PP1α antibody as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold alteration above control (± S.D.). (B) BAEC were treated as described in the legend of Fig. 2. PP1 activity was measured by serine/threonine phosphatase assay kit (Promega), quantified by measuring the fluorescence intensity (at 485/530 nm) using FACSCalibur flow cytometer (BD), and normalized to the fluorescence intensity from the control experiment. The bar graph shows the mean fold decreases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-bt-22-510: Arsenite decreases PP1 activity and NAC reverses arsenite-induced decrease in PP1 activity. (A) BAEC were treated as described in the legends of Fig. 1. The level of PP1 was measured by Western blot analysis using anti-PP1α antibody as described in the legend of Fig. 1. The blots are representative and the bar graph shows the mean fold alteration above control (± S.D.). (B) BAEC were treated as described in the legend of Fig. 2. PP1 activity was measured by serine/threonine phosphatase assay kit (Promega), quantified by measuring the fluorescence intensity (at 485/530 nm) using FACSCalibur flow cytometer (BD), and normalized to the fluorescence intensity from the control experiment. The bar graph shows the mean fold decreases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05.
Mentions: To elucidate the molecular mechanism by which arsenite acutely increases PP1-mediated eNOS-Thr497 phosphorylation, we investigated whether arsenite indeed decreases the protein level and/or the activity of PP1 in BAEC. Although it was reported that arsenite treatment decreased the level of PP1α expression in human lymphoblastoid cells (Tapio et al., 2005), we failed to find decrease in the level of PP1α expression in arsenite-treated BAEC (Fig. 4A). Instead, arsenite significantly decreased PP1 activity by ∼15% (Fig. 4B). Furthermore, the arsenite-induced decrease in PP1 activity was almost completely recovered by the pretreatment of NAC, suggesting that ROS mediates decreased PP1 activity 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