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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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Hydrogen peroxide inhibits vWF release in vivo. (A) The catalase inhibitor 3-AT increases H2O2 in cells. HAEC were pretreated with 3-AT for 2 h. 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. 0 mM). (B) The catalase inhibitor 3-AT decreases thrombin-induced exocytosis. HAEC were pretreated with 3-AT for 2 h, and then incubated with thrombin for 1 h. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 vs. thrombin alone). (C) The catalase inhibitor 3-AT increases H2O2 in mice. Mice were pretreated with 500 mg/kg 3-AT i.p. After 5 h, the amount of H2O2 released from spleen homogenates was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; *, P < 0.01 vs. control). (D) The catalase inhibitor 3-AT decreases platelet rolling in mice. Mice were pretreated or not with 500 mg/kg 3-AT, and after 5 h the mice were anesthetized and injected with calcein-AM–labeled platelets from nontreated mice. The mesentery was externalized, and 120–150-μm-diam venules were treated with 1 mM histamine. Platelet rolling on venules was imaged with a digital fluorescent camera (n = 5–6 ± SEM). (E) Digital fluorescence images of platelet rolling in mice treated with control or 3-AT (representative images from n = 5–6).
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fig6: Hydrogen peroxide inhibits vWF release in vivo. (A) The catalase inhibitor 3-AT increases H2O2 in cells. HAEC were pretreated with 3-AT for 2 h. 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. 0 mM). (B) The catalase inhibitor 3-AT decreases thrombin-induced exocytosis. HAEC were pretreated with 3-AT for 2 h, and then incubated with thrombin for 1 h. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 vs. thrombin alone). (C) The catalase inhibitor 3-AT increases H2O2 in mice. Mice were pretreated with 500 mg/kg 3-AT i.p. After 5 h, the amount of H2O2 released from spleen homogenates was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; *, P < 0.01 vs. control). (D) The catalase inhibitor 3-AT decreases platelet rolling in mice. Mice were pretreated or not with 500 mg/kg 3-AT, and after 5 h the mice were anesthetized and injected with calcein-AM–labeled platelets from nontreated mice. The mesentery was externalized, and 120–150-μm-diam venules were treated with 1 mM histamine. Platelet rolling on venules was imaged with a digital fluorescent camera (n = 5–6 ± SEM). (E) Digital fluorescence images of platelet rolling in mice treated with control or 3-AT (representative images from n = 5–6).

Mentions: We also examined the physiological effects of H2O2 on exocytosis in vivo. If H2O2 inhibits exocytosis, then we would expect catalase inhibitors to increase endogenous H2O2 levels and to decrease endothelial release of vWF. We first tested this hypothesis in endothelial cells with the catalase inhibitor 3-amino-triazole (3-AT). Increasing doses of 3-AT increase endothelial levels of H2O2 (Fig. 6 A). Increasing doses of 3-AT also block endothelial exocytosis (Fig. 6 B). We examined this phenomenon in mice. We administered 3-AT to mice, and examined H2O2 levels and exocytosis after 5 h. The catalase inhibitor 3-AT increases H2O2 levels in murine liver (Fig. 6 C). We examined the effect of 3-AT on platelet rolling along murine venules stimulated with FeCl3; platelet rolling is mediated in part by vWF released by endothelial exocytosis of Weibel-Palade bodies (Andre et al., 2000). Mice were treated with 3-AT or PBS, anesthetized, and injected with calcein-AM–labeled platelets. The mesentery was externalized, endothelial exocytosis was induced by superfusing with FeCl3, and platelet rolling on mesenteric venules was recorded using a digital fluorescent camera. FeCl3 activates platelet rolling in control mice (Fig. 6, D and E). However, 3-AT greatly inhibits FeCl3-activated platelet rolling in mice (Fig. 6, D and E). Together, these data suggest that H2O2 regulates endothelial exocytosis in vivo.


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)

Hydrogen peroxide inhibits vWF release in vivo. (A) The catalase inhibitor 3-AT increases H2O2 in cells. HAEC were pretreated with 3-AT for 2 h. 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. 0 mM). (B) The catalase inhibitor 3-AT decreases thrombin-induced exocytosis. HAEC were pretreated with 3-AT for 2 h, and then incubated with thrombin for 1 h. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 vs. thrombin alone). (C) The catalase inhibitor 3-AT increases H2O2 in mice. Mice were pretreated with 500 mg/kg 3-AT i.p. After 5 h, the amount of H2O2 released from spleen homogenates was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; *, P < 0.01 vs. control). (D) The catalase inhibitor 3-AT decreases platelet rolling in mice. Mice were pretreated or not with 500 mg/kg 3-AT, and after 5 h the mice were anesthetized and injected with calcein-AM–labeled platelets from nontreated mice. The mesentery was externalized, and 120–150-μm-diam venules were treated with 1 mM histamine. Platelet rolling on venules was imaged with a digital fluorescent camera (n = 5–6 ± SEM). (E) Digital fluorescence images of platelet rolling in mice treated with control or 3-AT (representative images from n = 5–6).
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fig6: Hydrogen peroxide inhibits vWF release in vivo. (A) The catalase inhibitor 3-AT increases H2O2 in cells. HAEC were pretreated with 3-AT for 2 h. 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. 0 mM). (B) The catalase inhibitor 3-AT decreases thrombin-induced exocytosis. HAEC were pretreated with 3-AT for 2 h, and then incubated with thrombin for 1 h. The amount of vWF released from cells into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.01 vs. thrombin alone). (C) The catalase inhibitor 3-AT increases H2O2 in mice. Mice were pretreated with 500 mg/kg 3-AT i.p. After 5 h, the amount of H2O2 released from spleen homogenates was measured by monitoring the increase in fluorescence of N-acetyl-3,7-dihydroxyphenoxazine (n = 3 ± SD; *, P < 0.01 vs. control). (D) The catalase inhibitor 3-AT decreases platelet rolling in mice. Mice were pretreated or not with 500 mg/kg 3-AT, and after 5 h the mice were anesthetized and injected with calcein-AM–labeled platelets from nontreated mice. The mesentery was externalized, and 120–150-μm-diam venules were treated with 1 mM histamine. Platelet rolling on venules was imaged with a digital fluorescent camera (n = 5–6 ± SEM). (E) Digital fluorescence images of platelet rolling in mice treated with control or 3-AT (representative images from n = 5–6).
Mentions: We also examined the physiological effects of H2O2 on exocytosis in vivo. If H2O2 inhibits exocytosis, then we would expect catalase inhibitors to increase endogenous H2O2 levels and to decrease endothelial release of vWF. We first tested this hypothesis in endothelial cells with the catalase inhibitor 3-amino-triazole (3-AT). Increasing doses of 3-AT increase endothelial levels of H2O2 (Fig. 6 A). Increasing doses of 3-AT also block endothelial exocytosis (Fig. 6 B). We examined this phenomenon in mice. We administered 3-AT to mice, and examined H2O2 levels and exocytosis after 5 h. The catalase inhibitor 3-AT increases H2O2 levels in murine liver (Fig. 6 C). We examined the effect of 3-AT on platelet rolling along murine venules stimulated with FeCl3; platelet rolling is mediated in part by vWF released by endothelial exocytosis of Weibel-Palade bodies (Andre et al., 2000). Mice were treated with 3-AT or PBS, anesthetized, and injected with calcein-AM–labeled platelets. The mesentery was externalized, endothelial exocytosis was induced by superfusing with FeCl3, and platelet rolling on mesenteric venules was recorded using a digital fluorescent camera. FeCl3 activates platelet rolling in control mice (Fig. 6, D and E). However, 3-AT greatly inhibits FeCl3-activated platelet rolling in mice (Fig. 6, D and E). Together, these data suggest that H2O2 regulates endothelial exocytosis in vivo.

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