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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

PKC is not involved in arsenite-induced eNOS-Thr497 phosphorylation, but calyculin A mimics the effect of arsenite on eNOS-Thr497 phosphorylation. BAEC were pretreated with (A) 14 (+) or 28 μM (++) Ro318425 for 0.5 h and then treated with 30 μM sodium arsenite for 4 h. Control cells were treated with vehicle only. The blots shown are representative of at least three experiments. (B) BAEC, transfected with HA-tagged cDNA encoding dominant negative (DN) conventional (α, βI, or βII), novel (δ or ε), or atypical (ζ) PKC gene, were treated with vehicle or 30 μM sodium arsenite for 4 h. Overexpression of the PKC gene after transfection was confirmed by detecting the tagged-HA. The blots shown are representative of at least three experiments. In separate experiments, BAEC were treated with (C) 2.5 or 5 nM okadaic acid for 0.5 h, or (D) 1, 2.5 or 5 nM calyculin A for 0.5 h. Control cells were treated with vehicle (DMSO) alone. (E) In some experiments, cells were pretreated with 5 nM calyculin A, vehicle, or 5 nM okadaic acid for 0.5 h and then 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 shown are representative of at least three experiments. (A-E) The bar graph shows the mean fold increases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. ns, not significant.
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f3-bt-22-510: PKC is not involved in arsenite-induced eNOS-Thr497 phosphorylation, but calyculin A mimics the effect of arsenite on eNOS-Thr497 phosphorylation. BAEC were pretreated with (A) 14 (+) or 28 μM (++) Ro318425 for 0.5 h and then treated with 30 μM sodium arsenite for 4 h. Control cells were treated with vehicle only. The blots shown are representative of at least three experiments. (B) BAEC, transfected with HA-tagged cDNA encoding dominant negative (DN) conventional (α, βI, or βII), novel (δ or ε), or atypical (ζ) PKC gene, were treated with vehicle or 30 μM sodium arsenite for 4 h. Overexpression of the PKC gene after transfection was confirmed by detecting the tagged-HA. The blots shown are representative of at least three experiments. In separate experiments, BAEC were treated with (C) 2.5 or 5 nM okadaic acid for 0.5 h, or (D) 1, 2.5 or 5 nM calyculin A for 0.5 h. Control cells were treated with vehicle (DMSO) alone. (E) In some experiments, cells were pretreated with 5 nM calyculin A, vehicle, or 5 nM okadaic acid for 0.5 h and then 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 shown are representative of at least three experiments. (A-E) The bar graph shows the mean fold increases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. ns, not significant.

Mentions: PKC has been reported to phosphorylate eNOS-Thr497 in in vitro phosphorylation experiment (Matsubara et al., 2003) and in cultured EC (Fleming et al., 2001; Matsubara et al., 2003). These data, together with previous report that ROS had also been to be capable of activating PKC through oxidation of its N-terminal regulatory domain (Cosentino-Gomes et al., 2012), prompted us to examine whether PKC mediates the arsenite-induced increase in eNOS-Thr497 phosphorylation. Experiment evaluating the effect of PKC-specific inhibitor, Ro318425, however, did not alter the arsenite-stimulated eNOS-Thr497 phosphorylation (Fig. 3A). To further clarify these data, we transfected dominant-negative (DN) PKC isoforms, α, βI, βII, δ, ε and ζ, into BAEC. In accordance with the result from PKC inhibitor experiment, overexpression of DN-PKC genes did not reverse the increased eNOS-Thr497 phosphorylation by arsenite (Fig. 3B), which suggests that PKC is not involved in the arsenite-stimulated increase in eNOS-Thr497 phosphorylation.


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)

PKC is not involved in arsenite-induced eNOS-Thr497 phosphorylation, but calyculin A mimics the effect of arsenite on eNOS-Thr497 phosphorylation. BAEC were pretreated with (A) 14 (+) or 28 μM (++) Ro318425 for 0.5 h and then treated with 30 μM sodium arsenite for 4 h. Control cells were treated with vehicle only. The blots shown are representative of at least three experiments. (B) BAEC, transfected with HA-tagged cDNA encoding dominant negative (DN) conventional (α, βI, or βII), novel (δ or ε), or atypical (ζ) PKC gene, were treated with vehicle or 30 μM sodium arsenite for 4 h. Overexpression of the PKC gene after transfection was confirmed by detecting the tagged-HA. The blots shown are representative of at least three experiments. In separate experiments, BAEC were treated with (C) 2.5 or 5 nM okadaic acid for 0.5 h, or (D) 1, 2.5 or 5 nM calyculin A for 0.5 h. Control cells were treated with vehicle (DMSO) alone. (E) In some experiments, cells were pretreated with 5 nM calyculin A, vehicle, or 5 nM okadaic acid for 0.5 h and then 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 shown are representative of at least three experiments. (A-E) The bar graph shows the mean fold increases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3-bt-22-510: PKC is not involved in arsenite-induced eNOS-Thr497 phosphorylation, but calyculin A mimics the effect of arsenite on eNOS-Thr497 phosphorylation. BAEC were pretreated with (A) 14 (+) or 28 μM (++) Ro318425 for 0.5 h and then treated with 30 μM sodium arsenite for 4 h. Control cells were treated with vehicle only. The blots shown are representative of at least three experiments. (B) BAEC, transfected with HA-tagged cDNA encoding dominant negative (DN) conventional (α, βI, or βII), novel (δ or ε), or atypical (ζ) PKC gene, were treated with vehicle or 30 μM sodium arsenite for 4 h. Overexpression of the PKC gene after transfection was confirmed by detecting the tagged-HA. The blots shown are representative of at least three experiments. In separate experiments, BAEC were treated with (C) 2.5 or 5 nM okadaic acid for 0.5 h, or (D) 1, 2.5 or 5 nM calyculin A for 0.5 h. Control cells were treated with vehicle (DMSO) alone. (E) In some experiments, cells were pretreated with 5 nM calyculin A, vehicle, or 5 nM okadaic acid for 0.5 h and then 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 shown are representative of at least three experiments. (A-E) The bar graph shows the mean fold increases above control (± S.D.) (n=3). Differences were statistically significant at *p<0.05 and **p<0.01. ns, not significant.
Mentions: PKC has been reported to phosphorylate eNOS-Thr497 in in vitro phosphorylation experiment (Matsubara et al., 2003) and in cultured EC (Fleming et al., 2001; Matsubara et al., 2003). These data, together with previous report that ROS had also been to be capable of activating PKC through oxidation of its N-terminal regulatory domain (Cosentino-Gomes et al., 2012), prompted us to examine whether PKC mediates the arsenite-induced increase in eNOS-Thr497 phosphorylation. Experiment evaluating the effect of PKC-specific inhibitor, Ro318425, however, did not alter the arsenite-stimulated eNOS-Thr497 phosphorylation (Fig. 3A). To further clarify these data, we transfected dominant-negative (DN) PKC isoforms, α, βI, βII, δ, ε and ζ, into BAEC. In accordance with the result from PKC inhibitor experiment, overexpression of DN-PKC genes did not reverse the increased eNOS-Thr497 phosphorylation by arsenite (Fig. 3B), which suggests that PKC is not involved in the arsenite-stimulated increase in eNOS-Thr497 phosphorylation.

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