Limits...
Hydrogen peroxide regulation of endothelial exocytosis by inhibition of N-ethylmaleimide sensitive factor.

Matsushita K, Morrell CN, Mason RJ, Yamakuchi M, Khanday FA, Irani K, Lowenstein CJ - J. Cell Biol. (2005)

Bottom Line: H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex.Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF.Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Although an excess of reactive oxygen species (ROS) can damage the vasculature, low concentrations of ROS mediate intracellular signal transduction pathways. We hypothesized that hydrogen peroxide plays a beneficial role in the vasculature by inhibiting endothelial exocytosis that would otherwise induce vascular inflammation and thrombosis. We now show that endogenous H(2)O(2) inhibits thrombin-induced exocytosis of granules from endothelial cells. H(2)O(2) regulates exocytosis by inhibiting N-ethylmaleimide sensitive factor (NSF), a protein that regulates membrane fusion events necessary for exocytosis. H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex. Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF. Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo. By inhibiting endothelial cell exocytosis, endogenous H(2)O(2) may protect the vasculature from inflammation and thrombosis.

Show MeSH

Related in: MedlinePlus

Exogenous H2O2 inhibits exocytosis from HAEC. (A) Dose response. HAEC were pretreated with increasing amounts of exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.05 vs. thrombin; **, P < 0.01 vs. thrombin). (B) Time course. HAEC were pretreated with exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media at increasing times was measured by an ELISA (n = 3 ± SD). (C) NAC decreases exogenous H2O2 inhibition of thrombin-triggered vWF release. Cells were incubated with 10 mM NAC for 4 h, washed, treated with H2O2, and stimulated with media or thrombin, and the amount of released vWF was measured (n = 3 ± SD; **, P < 0.01 vs. H2O2 alone).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171382&req=5

fig1: Exogenous H2O2 inhibits exocytosis from HAEC. (A) Dose response. HAEC were pretreated with increasing amounts of exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.05 vs. thrombin; **, P < 0.01 vs. thrombin). (B) Time course. HAEC were pretreated with exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media at increasing times was measured by an ELISA (n = 3 ± SD). (C) NAC decreases exogenous H2O2 inhibition of thrombin-triggered vWF release. Cells were incubated with 10 mM NAC for 4 h, washed, treated with H2O2, and stimulated with media or thrombin, and the amount of released vWF was measured (n = 3 ± SD; **, P < 0.01 vs. H2O2 alone).

Mentions: To explore the effect of H2O2 on granule exocytosis, we studied thrombin-induced exocytosis of Weibel-Palade bodies from human aortic endothelial cells (HAEC). We pretreated HAEC with H2O2 for 10 min, and then stimulated the cells with thrombin, and measured the amount of vWF released into the media. Thrombin induces the rapid release of vWF from HAEC (Fig. 1, A and B). However, exogenous H2O2 blocks the effects of thrombin in a dose-dependent manner (Fig. 1, A and B). We next examined the effect of the antioxidant N-acetyl-cysteine (NAC) on Weibel-Palade body exocytosis. HAEC were incubated with NAC for 4 h, washed, treated with H2O2, and finally stimulated with media or thrombin, and exocytosis was measured with an ELISA for vWF. NAC counteracts the inhibitory effect of H2O2 (Fig. 1 C).


Hydrogen peroxide regulation of endothelial exocytosis by inhibition of N-ethylmaleimide sensitive factor.

Matsushita K, Morrell CN, Mason RJ, Yamakuchi M, Khanday FA, Irani K, Lowenstein CJ - J. Cell Biol. (2005)

Exogenous H2O2 inhibits exocytosis from HAEC. (A) Dose response. HAEC were pretreated with increasing amounts of exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.05 vs. thrombin; **, P < 0.01 vs. thrombin). (B) Time course. HAEC were pretreated with exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media at increasing times was measured by an ELISA (n = 3 ± SD). (C) NAC decreases exogenous H2O2 inhibition of thrombin-triggered vWF release. Cells were incubated with 10 mM NAC for 4 h, washed, treated with H2O2, and stimulated with media or thrombin, and the amount of released vWF was measured (n = 3 ± SD; **, P < 0.01 vs. H2O2 alone).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171382&req=5

fig1: Exogenous H2O2 inhibits exocytosis from HAEC. (A) Dose response. HAEC were pretreated with increasing amounts of exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.05 vs. thrombin; **, P < 0.01 vs. thrombin). (B) Time course. HAEC were pretreated with exogenous H2O2 for 10 min, and then treated with 1 U/ml thrombin, and the amount of vWF released into the media at increasing times was measured by an ELISA (n = 3 ± SD). (C) NAC decreases exogenous H2O2 inhibition of thrombin-triggered vWF release. Cells were incubated with 10 mM NAC for 4 h, washed, treated with H2O2, and stimulated with media or thrombin, and the amount of released vWF was measured (n = 3 ± SD; **, P < 0.01 vs. H2O2 alone).
Mentions: To explore the effect of H2O2 on granule exocytosis, we studied thrombin-induced exocytosis of Weibel-Palade bodies from human aortic endothelial cells (HAEC). We pretreated HAEC with H2O2 for 10 min, and then stimulated the cells with thrombin, and measured the amount of vWF released into the media. Thrombin induces the rapid release of vWF from HAEC (Fig. 1, A and B). However, exogenous H2O2 blocks the effects of thrombin in a dose-dependent manner (Fig. 1, A and B). We next examined the effect of the antioxidant N-acetyl-cysteine (NAC) on Weibel-Palade body exocytosis. HAEC were incubated with NAC for 4 h, washed, treated with H2O2, and finally stimulated with media or thrombin, and exocytosis was measured with an ELISA for vWF. NAC counteracts the inhibitory effect of H2O2 (Fig. 1 C).

Bottom Line: H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex.Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF.Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Although an excess of reactive oxygen species (ROS) can damage the vasculature, low concentrations of ROS mediate intracellular signal transduction pathways. We hypothesized that hydrogen peroxide plays a beneficial role in the vasculature by inhibiting endothelial exocytosis that would otherwise induce vascular inflammation and thrombosis. We now show that endogenous H(2)O(2) inhibits thrombin-induced exocytosis of granules from endothelial cells. H(2)O(2) regulates exocytosis by inhibiting N-ethylmaleimide sensitive factor (NSF), a protein that regulates membrane fusion events necessary for exocytosis. H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex. Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF. Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo. By inhibiting endothelial cell exocytosis, endogenous H(2)O(2) may protect the vasculature from inflammation and thrombosis.

Show MeSH
Related in: MedlinePlus