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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.

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Endogenous H2O2 inhibits exocytosis from HAEC. (A) Catalase inhibits endogenous H2O2 production. HAEC were transduced with adenoviral vectors and stimulated with thrombin. The amount of H2O2 released from cells was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; **, P < 0.01 vs. control). (B) Endogenous H2O2 decreases thrombin-triggered vWF release. HAEC were transduced with adenoviral vectors and, after 48 h, treated with thrombin, and vWF was measured with an ELISA (n = 3 ± SD; **, P < 0.01 vs. none). (C) Angiotensin II induction of endogenous H2O2 decreases vWF release. HAEC were stimulated with 10−7 M angiotensin II for 30 min, and then incubated with thrombin and 500 U/ml catalase or 10 mM angiotensin II antipeptide. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; **, P < 0.01 vs. control).
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fig2: Endogenous H2O2 inhibits exocytosis from HAEC. (A) Catalase inhibits endogenous H2O2 production. HAEC were transduced with adenoviral vectors and stimulated with thrombin. The amount of H2O2 released from cells was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; **, P < 0.01 vs. control). (B) Endogenous H2O2 decreases thrombin-triggered vWF release. HAEC were transduced with adenoviral vectors and, after 48 h, treated with thrombin, and vWF was measured with an ELISA (n = 3 ± SD; **, P < 0.01 vs. none). (C) Angiotensin II induction of endogenous H2O2 decreases vWF release. HAEC were stimulated with 10−7 M angiotensin II for 30 min, and then incubated with thrombin and 500 U/ml catalase or 10 mM angiotensin II antipeptide. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; **, P < 0.01 vs. control).

Mentions: We next examined the role of endogenous H2O2 in the regulation of Weibel-Palade body exocytosis. First, we showed that thrombin increases endogenous H2O2 production. We transduced HAEC with adenoviral vectors expressing β-galactosidase or catalase and measured cellular levels of H2O2 before and after thrombin treatment. Thrombin increases endogenous H2O2 production in control cells, but transduction with adenovirus-catalase blocks thrombin stimulation of endogenous H2O2 production (Fig. 2 A). These data suggest that thrombin activates endogenous H2O2 production and catalase decreases endogenous H2O2 levels.


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)

Endogenous H2O2 inhibits exocytosis from HAEC. (A) Catalase inhibits endogenous H2O2 production. HAEC were transduced with adenoviral vectors and stimulated with thrombin. The amount of H2O2 released from cells was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; **, P < 0.01 vs. control). (B) Endogenous H2O2 decreases thrombin-triggered vWF release. HAEC were transduced with adenoviral vectors and, after 48 h, treated with thrombin, and vWF was measured with an ELISA (n = 3 ± SD; **, P < 0.01 vs. none). (C) Angiotensin II induction of endogenous H2O2 decreases vWF release. HAEC were stimulated with 10−7 M angiotensin II for 30 min, and then incubated with thrombin and 500 U/ml catalase or 10 mM angiotensin II antipeptide. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; **, P < 0.01 vs. control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171382&req=5

fig2: Endogenous H2O2 inhibits exocytosis from HAEC. (A) Catalase inhibits endogenous H2O2 production. HAEC were transduced with adenoviral vectors and stimulated with thrombin. The amount of H2O2 released from cells was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; **, P < 0.01 vs. control). (B) Endogenous H2O2 decreases thrombin-triggered vWF release. HAEC were transduced with adenoviral vectors and, after 48 h, treated with thrombin, and vWF was measured with an ELISA (n = 3 ± SD; **, P < 0.01 vs. none). (C) Angiotensin II induction of endogenous H2O2 decreases vWF release. HAEC were stimulated with 10−7 M angiotensin II for 30 min, and then incubated with thrombin and 500 U/ml catalase or 10 mM angiotensin II antipeptide. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; **, P < 0.01 vs. control).
Mentions: We next examined the role of endogenous H2O2 in the regulation of Weibel-Palade body exocytosis. First, we showed that thrombin increases endogenous H2O2 production. We transduced HAEC with adenoviral vectors expressing β-galactosidase or catalase and measured cellular levels of H2O2 before and after thrombin treatment. Thrombin increases endogenous H2O2 production in control cells, but transduction with adenovirus-catalase blocks thrombin stimulation of endogenous H2O2 production (Fig. 2 A). These data suggest that thrombin activates endogenous H2O2 production and catalase decreases endogenous H2O2 levels.

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