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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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H2O2 inhibits NSF. (A) H2O2 inhibits ATPase activity of wild-type NSF. H2O2 or control was added to recombinant wild-type NSF, and the ATPase activity of NSF was measured (n = 2 ± SD; *, P < 0.05 vs. NSF; **, P < 0.01 vs. NSF). (B) DTT restores ATPase activity of wild-type NSF inhibited by H2O2. H2O2 or control was added to recombinant wild-type NSF, buffer or 1 mM DTT was added, and the ATPase activity of NSF was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + DTT). (C) H2O2 inhibits disassembly activity of wild-type NSF. Recombinant (His)6-NSF was pretreated or not with 1 mM H2O2 and incubated with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. ATP or ATP-γS was added, and the mixture was precipitated with glutathione-sepharose. 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. (D) H2O2 inhibits disassembly activity of wild-type NSF (dose response). The NSF disassembly assay was performed, pretreating recombinant (His)6-NSF with increasing concentrations of H2O2 and then mixing with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. Proteins precipitated with glutathione-sepharose 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. (E) Exogenous NSF restores vWF exocytosis in endothelial cells treated with H2O2. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized with SLO, incubated with recombinant NSF or H2O2-treated recombinant NSF, and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + NSF). (F) NSF mutant C264A does not restore exocytosis. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized, and incubated with recombinant wild-type NSF (WT) or mutant NSF(C264A). In some cases, the recombinant NSF was treated with H2O2 before addition to cells. The cells were then resealed and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 vs. H2O2 + Thrombin).
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fig3: H2O2 inhibits NSF. (A) H2O2 inhibits ATPase activity of wild-type NSF. H2O2 or control was added to recombinant wild-type NSF, and the ATPase activity of NSF was measured (n = 2 ± SD; *, P < 0.05 vs. NSF; **, P < 0.01 vs. NSF). (B) DTT restores ATPase activity of wild-type NSF inhibited by H2O2. H2O2 or control was added to recombinant wild-type NSF, buffer or 1 mM DTT was added, and the ATPase activity of NSF was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + DTT). (C) H2O2 inhibits disassembly activity of wild-type NSF. Recombinant (His)6-NSF was pretreated or not with 1 mM H2O2 and incubated with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. ATP or ATP-γS was added, and the mixture was precipitated with glutathione-sepharose. 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. (D) H2O2 inhibits disassembly activity of wild-type NSF (dose response). The NSF disassembly assay was performed, pretreating recombinant (His)6-NSF with increasing concentrations of H2O2 and then mixing with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. Proteins precipitated with glutathione-sepharose 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. (E) Exogenous NSF restores vWF exocytosis in endothelial cells treated with H2O2. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized with SLO, incubated with recombinant NSF or H2O2-treated recombinant NSF, and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + NSF). (F) NSF mutant C264A does not restore exocytosis. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized, and incubated with recombinant wild-type NSF (WT) or mutant NSF(C264A). In some cases, the recombinant NSF was treated with H2O2 before addition to cells. The cells were then resealed and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 vs. H2O2 + Thrombin).

Mentions: How does H2O2 inhibit exocytosis? We hypothesized that H2O2 inhibits NSF, a protein that regulates granule exocytosis, by hydrolyzing ATP and by interacting with SNARE molecules (Block et al., 1988; Malhotra et al., 1988; Mellman and Warren, 2000). We first examined the effect of H2O2 on the ATPase activity of NSF, which is critical for NSF function (Whiteheart et al., 1994). H2O2 was added to 10 μg of recombinant NSF, and the ATPase activity of NSF was measured by a colorimetric assay. H2O2 significantly inhibits NSF hydrolysis of ATP (Fig. 3 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)

H2O2 inhibits NSF. (A) H2O2 inhibits ATPase activity of wild-type NSF. H2O2 or control was added to recombinant wild-type NSF, and the ATPase activity of NSF was measured (n = 2 ± SD; *, P < 0.05 vs. NSF; **, P < 0.01 vs. NSF). (B) DTT restores ATPase activity of wild-type NSF inhibited by H2O2. H2O2 or control was added to recombinant wild-type NSF, buffer or 1 mM DTT was added, and the ATPase activity of NSF was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + DTT). (C) H2O2 inhibits disassembly activity of wild-type NSF. Recombinant (His)6-NSF was pretreated or not with 1 mM H2O2 and incubated with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. ATP or ATP-γS was added, and the mixture was precipitated with glutathione-sepharose. 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. (D) H2O2 inhibits disassembly activity of wild-type NSF (dose response). The NSF disassembly assay was performed, pretreating recombinant (His)6-NSF with increasing concentrations of H2O2 and then mixing with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. Proteins precipitated with glutathione-sepharose 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. (E) Exogenous NSF restores vWF exocytosis in endothelial cells treated with H2O2. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized with SLO, incubated with recombinant NSF or H2O2-treated recombinant NSF, and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + NSF). (F) NSF mutant C264A does not restore exocytosis. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized, and incubated with recombinant wild-type NSF (WT) or mutant NSF(C264A). In some cases, the recombinant NSF was treated with H2O2 before addition to cells. The cells were then resealed and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 vs. H2O2 + Thrombin).
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fig3: H2O2 inhibits NSF. (A) H2O2 inhibits ATPase activity of wild-type NSF. H2O2 or control was added to recombinant wild-type NSF, and the ATPase activity of NSF was measured (n = 2 ± SD; *, P < 0.05 vs. NSF; **, P < 0.01 vs. NSF). (B) DTT restores ATPase activity of wild-type NSF inhibited by H2O2. H2O2 or control was added to recombinant wild-type NSF, buffer or 1 mM DTT was added, and the ATPase activity of NSF was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + DTT). (C) H2O2 inhibits disassembly activity of wild-type NSF. Recombinant (His)6-NSF was pretreated or not with 1 mM H2O2 and incubated with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. ATP or ATP-γS was added, and the mixture was precipitated with glutathione-sepharose. 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. (D) H2O2 inhibits disassembly activity of wild-type NSF (dose response). The NSF disassembly assay was performed, pretreating recombinant (His)6-NSF with increasing concentrations of H2O2 and then mixing with (His)6-α-SNAP, GST-Syntaxin-4, VAMP-3, and SNAP-23. Proteins precipitated with glutathione-sepharose 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. (E) Exogenous NSF restores vWF exocytosis in endothelial cells treated with H2O2. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized with SLO, incubated with recombinant NSF or H2O2-treated recombinant NSF, and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 for H2O2 vs. H2O2 + NSF). (F) NSF mutant C264A does not restore exocytosis. HAEC were pretreated with 1 mM H2O2 for 10 min, permeabilized, and incubated with recombinant wild-type NSF (WT) or mutant NSF(C264A). In some cases, the recombinant NSF was treated with H2O2 before addition to cells. The cells were then resealed and stimulated with thrombin, and the amount of vWF in the media was measured (n = 3 ± SD; *, P < 0.01 vs. H2O2 + Thrombin).
Mentions: How does H2O2 inhibit exocytosis? We hypothesized that H2O2 inhibits NSF, a protein that regulates granule exocytosis, by hydrolyzing ATP and by interacting with SNARE molecules (Block et al., 1988; Malhotra et al., 1988; Mellman and Warren, 2000). We first examined the effect of H2O2 on the ATPase activity of NSF, which is critical for NSF function (Whiteheart et al., 1994). H2O2 was added to 10 μg of recombinant NSF, and the ATPase activity of NSF was measured by a colorimetric assay. H2O2 significantly inhibits NSF hydrolysis of ATP (Fig. 3 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