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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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Mutant NSF(C264T) is resistant to H2O2. (A) H2O2 does not affect ATPase activity of mutant NSF(C264T). The ATPase assay was performed, adding recombinant wild-type or mutant NSF. 100 μM H2O2 inhibits ATPase activity of wild-type NSF but not of mutant NSF(C264T). H2O2 has no effect on ATPase activity of NSF(C264A) (n = 3 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect disassembly activity of specific NSF mutants. The disassembly assay was performed, adding α-SNAP, GST-Syntaxin-4, VAMP-3, SNAP-23, and recombinant wild-type NSF (WT) or mutant NSF(C264A) or NSF(C264T). Precipitated proteins were immunoblotted with antibody to the NSF tag (top), to syntaxin-4 (middle), or to VAMP-3 (bottom). Experiment was repeated three times with similar results. 1 mM H2O2 inhibits disassembly activity of wild-type NSF but not of mutant NSF(C264T). (C) H2O2 does not affect exocytosis in endothelial cells containing NSF mutants. HAEC were permeabilized with SLO and incubated with recombinant wild-type NSF or mutant NSF(C264T). Cells were resealed, pretreated with 0 or 100 μM H2O2 for 10 min, and treated with thrombin. The amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 for thrombin vs. thrombin + H2O2).
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fig5: Mutant NSF(C264T) is resistant to H2O2. (A) H2O2 does not affect ATPase activity of mutant NSF(C264T). The ATPase assay was performed, adding recombinant wild-type or mutant NSF. 100 μM H2O2 inhibits ATPase activity of wild-type NSF but not of mutant NSF(C264T). H2O2 has no effect on ATPase activity of NSF(C264A) (n = 3 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect disassembly activity of specific NSF mutants. The disassembly assay was performed, adding α-SNAP, GST-Syntaxin-4, VAMP-3, SNAP-23, and recombinant wild-type NSF (WT) or mutant NSF(C264A) or NSF(C264T). Precipitated proteins were immunoblotted with antibody to the NSF tag (top), to syntaxin-4 (middle), or to VAMP-3 (bottom). Experiment was repeated three times with similar results. 1 mM H2O2 inhibits disassembly activity of wild-type NSF but not of mutant NSF(C264T). (C) H2O2 does not affect exocytosis in endothelial cells containing NSF mutants. HAEC were permeabilized with SLO and incubated with recombinant wild-type NSF or mutant NSF(C264T). Cells were resealed, pretreated with 0 or 100 μM H2O2 for 10 min, and treated with thrombin. The amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 for thrombin vs. thrombin + H2O2).

Mentions: We first compared the effect of H2O2 on the ATPase activity of recombinant wild-type NSF and mutant NSF(C264T). The ATPase activity of the NSF(C264T) mutant is approximately the same as that of wild-type NSF, although the ATPase activity of mutant NSF(C264A) is greatly decreased (Fig. 5 A). H2O2 inhibits ATPase activity of wild-type NSF but not of mutant NSF(C264T) (Fig. 5 A).


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)

Mutant NSF(C264T) is resistant to H2O2. (A) H2O2 does not affect ATPase activity of mutant NSF(C264T). The ATPase assay was performed, adding recombinant wild-type or mutant NSF. 100 μM H2O2 inhibits ATPase activity of wild-type NSF but not of mutant NSF(C264T). H2O2 has no effect on ATPase activity of NSF(C264A) (n = 3 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect disassembly activity of specific NSF mutants. The disassembly assay was performed, adding α-SNAP, GST-Syntaxin-4, VAMP-3, SNAP-23, and recombinant wild-type NSF (WT) or mutant NSF(C264A) or NSF(C264T). Precipitated proteins were immunoblotted with antibody to the NSF tag (top), to syntaxin-4 (middle), or to VAMP-3 (bottom). Experiment was repeated three times with similar results. 1 mM H2O2 inhibits disassembly activity of wild-type NSF but not of mutant NSF(C264T). (C) H2O2 does not affect exocytosis in endothelial cells containing NSF mutants. HAEC were permeabilized with SLO and incubated with recombinant wild-type NSF or mutant NSF(C264T). Cells were resealed, pretreated with 0 or 100 μM H2O2 for 10 min, and treated with thrombin. The amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 for thrombin vs. thrombin + H2O2).
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fig5: Mutant NSF(C264T) is resistant to H2O2. (A) H2O2 does not affect ATPase activity of mutant NSF(C264T). The ATPase assay was performed, adding recombinant wild-type or mutant NSF. 100 μM H2O2 inhibits ATPase activity of wild-type NSF but not of mutant NSF(C264T). H2O2 has no effect on ATPase activity of NSF(C264A) (n = 3 ± SD; **, P < 0.01 vs. control). (B) H2O2 does not affect disassembly activity of specific NSF mutants. The disassembly assay was performed, adding α-SNAP, GST-Syntaxin-4, VAMP-3, SNAP-23, and recombinant wild-type NSF (WT) or mutant NSF(C264A) or NSF(C264T). Precipitated proteins were immunoblotted with antibody to the NSF tag (top), to syntaxin-4 (middle), or to VAMP-3 (bottom). Experiment was repeated three times with similar results. 1 mM H2O2 inhibits disassembly activity of wild-type NSF but not of mutant NSF(C264T). (C) H2O2 does not affect exocytosis in endothelial cells containing NSF mutants. HAEC were permeabilized with SLO and incubated with recombinant wild-type NSF or mutant NSF(C264T). Cells were resealed, pretreated with 0 or 100 μM H2O2 for 10 min, and treated with thrombin. The amount of vWF released into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 for thrombin vs. thrombin + H2O2).
Mentions: We first compared the effect of H2O2 on the ATPase activity of recombinant wild-type NSF and mutant NSF(C264T). The ATPase activity of the NSF(C264T) mutant is approximately the same as that of wild-type NSF, although the ATPase activity of mutant NSF(C264A) is greatly decreased (Fig. 5 A). H2O2 inhibits ATPase activity of wild-type NSF but not of mutant NSF(C264T) (Fig. 5 A).

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