Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: Patient-derived ASPD increase eNOS-Thr 495 phosphorylation through a mitochondrial ROS-PKC pathway (A and B) Patient-derived ASPD were isolated from postmortem brains of the three AD patients according to the method established previously ( Noguchi et al., 2009 ; Ohnishi et al., 2015 ) (see “ ”). Primary human endothelial cells were pretreated with bisindolylmaleimide I (Bim I, 5 μM) or YCG-063 (YCG, 50 μM) for 30 min, and were further treated with patient-derived ASPD (1.6 nM) for 6 hr (see “ ”). The ratios of eNOS-P-Thr 495 to eNOS-total in A and PKC-P-Ser 660 to PKC-total in B were determined as in Figure 6 C (n = 4). Data are presented as means ± S.E. ∗ P < 0.05 (ANOVA with Scheffé’s method).

Article Snippet: Mouse anti-eNOS-P-Thr 495 antibody , BD Bioscience , Cat# 612707; RRID: AB_399946.

Techniques: Derivative Assay, Isolation

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: ASPD-NAKα3 interaction in caveolae increases the phosphorylation of eNOS-Thr 495 in primary human cerebral endothelial cells (A) Primary human brain microvessel endothelial cells, treated with ASPD at the indicated concentration for 0, 10, or 60 min, were multiply stained with the indicated antibodies; NAKα3-specific antibody, eNOS-specific antibody, lipid rafts flotillin-1-specific antibody, and ASPD-specific antibody (rabbit polyclonal rpASD1), along with DAPI nuclear stain, were used as described in “ ”. The weighted colocalization coefficients were obtained using ZEN2009 software (the eNOS-overlapped NAKα3/total NAKα3 ratios in the cells treated with ASPD for 0, 10, and 60 min are 55.7 ± 2.4%, 58.8 ± 4.8%, and 56.0 ± 2.4%, respectively; the NAKα3-overlapped eNOS/total eNOS ratios in the cells treated with ASPD for 0, 10, and 60 min are 40.8 ± 1.4%, 42.7 ± 2.1%, and 40.1 ± 1.7%, respectively; the flotillin-1-overlapped NAKα3/total NAKα3 ratios in the cells treated with ASPD for 0, 10, and 60 min are 4.5 ± 0.9%, 4.9 ± 1.2%, and 3.6 ± 0.9%, respectively: data are presented as means ± S.E. (n = 10)) (see “ ”). The weighted colocalization coefficients represent the number of red (or green) pixels that co-localize with green (or red) pixels divided by the total number of red (or green) pixels. Scale bars: 5 μm for solid line and 1 μm for hatched line. (B) Schematic illustration of the relationship between the NO production and the phosphorylation at Ser 1177 /Thr 495 of eNOS. Carbachol activates eNOS by inducing phosphorylation at Ser 1177 and dephosphorylation at Thr 495 in parallel (green arrows). ASPD increase Thr 495 phosphorylation of eNOS (red arrow) through an independent pathway from that of carbachol. (C) Primary human endothelial cells were treated with ASPD (35 nM) for 6 hr (see “ ”). The levels of eNOS-P-Ser 1177 , eNOS-P-Thr 495 , and eNOS-total were determined by Western blotting of total extracts with antibodies specific for eNOS-P-Ser 1177 , eNOS-P-Thr 495 , and eNOS-total, respectively, as shown in upper Western blots (see “ ”). Quantification data were determined by densitometry using LAS-4000 Mini software and are shown as the ratio of eNOS-P-Ser 1177 or eNOS-P-Thr 495 to eNOS-total. The ratio of non-treated cells is shown as 100 (n = 4). Data are presented as means ± S.E. (Welch’s t -test). (D) The endothelial cells were pretreated with ASPD (32 nM) for 6 hr and were further treated with carbachol (1 μM) for 5 min (see “ ”). The ratio of eNOS-P-Ser 1177 to eNOS-total was obtained and is shown as in C (n = 4). Data are presented as means ± S.E. ∗ P < 0.05/∗∗ P < 0.01 (ANOVA with Scheffé’s method). (E) The endothelial cells, with ATP1A3 siRNA or Mock siRNA transfection or without transfection as in Figure 3 E, were treated with ASPD (32 nM) for 6 hr. The ratio of eNOS-P-Thr 495 to eNOS-total was obtained as in C (n = 3). Data are presented as means ± S.E. ∗ P < 0.05/∗∗ P < 0.01 (ANOVA with Scheffé’s method).

Article Snippet: Mouse anti-eNOS-P-Thr 495 antibody , BD Bioscience , Cat# 612707; RRID: AB_399946.

Techniques: Concentration Assay, Staining, Software, De-Phosphorylation Assay, Western Blot, Transfection

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: The mitochondrial ROS/PKC pathway is involved in eNOS-Thr 495 phosphorylation by ASPD in primary human cerebral endothelial cells (A and B) Primary human endothelial cells were pretreated for 30 min either with bisindolylmaleimide I (Bim I, 5 μM), Y-27632 (Y27, 10 μM), or compound C (CC, 10 μM) in A or with calphostin C (Cal C, 0.3 μM) in B, and were further treated for 6 hr with ASPD (32 nM in A or 35 nM in B) (see “ ”). The ratio of eNOS-P-Thr 495 to eNOS-total was obtained as in Figure 5 C. The ratio in the non-treated cells is shown as 100 (n = 4 in A and 3 in B). As shown in Western blots in B, calphostin C decreased the eNOS-total without affecting cell survival, most likely due to its non-specific inhibition of phospholipase D activity ( Zheng et al., 2004 ). Nevertheless, because both bisindolylmaleimide I and calphostin C completely inhibited the ASPD-induced increase in eNOS-P-Thr 495 , this does not affect the conclusion that ASPD work through PKC activation. Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method). (C) The cells were treated with freshly dissolved Aβ 1-42 solution (1 μM) for 6 hr (see “ ”). The ratio of eNOS-P-Thr 495 to eNOS-total was determined as in A. The ratio of PKC-P-Ser 660 to PKC-total was determined by Western blotting of total extracts with antibodies specific for PKC-P-Ser 660 and PKC-total, respectively (n = 4). The results show that ASPD induce eNOS inactivation through PKC activation, whereas freshly dissolved Aβ 1-42 does not. Our findings also confirmed a possible link between Aβ and inactivation of eNOS, the molecular mechanism of which has been largely unknown, as previously reported ( Chisari et al., 2010 ; Gentile et al., 2004 ; Lamoke et al., 2015 ; Suhara et al., 2003 ). AMP kinase and Rho kinase have been also reported to play a role in the physiological regulation of eNOS by phosphorylating Thr 495 . These kinases might be involved in the case of Aβ 1-42 . Data are presented as means ± S.E. ∗ P < 0.05 (Welch's t test). (D and E) The cells were pretreated with tempol (Tem, 3 mM), BAPTA-AM (BAP, 30 μM), or U-73122 (U-73, 10 μM) for 30 min, and were further treated with ASPD (63 nM) for 6 hr (see “ ”). The ratios of eNOS-P-Thr 495 to eNOS-total and that of PKC-P-Ser 660 to PKC-total were obtained as above (n = 4). Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method). (F) The cells were pretreated with YCG-063 (YCG, 50 μM), mito-tempol (M-temp, 100 μM), VAS2870 (VAS, 10 μM), or apocynin (Apo, 20 μM) for 30 min (see “ ”), and were further treated with ASPD (35 nM) for 6 hr. ROS production was estimated by monitoring the fluorescence intensity of a ROS fluorescence indicator, CellROX (see “ ”) (n = 4). Representative fluorescence images, along with expanded images of the areas surrounded by hatched lines in the upper panels are shown. The CellROX fluorescence intensities were determined using a laser scanning cytometer CQ1 (see “ ”). Quantification data in ASPD-treated cells are shown as 100. Scale bars: 100 μm for solid line and 20 μm for hatched line. Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method).

Article Snippet: Mouse anti-eNOS-P-Thr 495 antibody , BD Bioscience , Cat# 612707; RRID: AB_399946.

Techniques: Western Blot, Inhibition, Activity Assay, Activation Assay, Fluorescence, Cytometry

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: Schematic illustration of the mechanism of ASPD-induced suppression of eNOS activity in brain microvessel endothelial cells ASPD bind to cell-surface NAKα3 in caveolae on cerebral microvessel endothelial cells, promote mitochondrial ROS production, activate PKC, increase eNOS-Thr 495 phosphorylation, and attenuate NO release, resulting in suppression of blood microvessel relaxation response.

Article Snippet: Mouse anti-eNOS-P-Thr 495 antibody , BD Bioscience , Cat# 612707; RRID: AB_399946.

Techniques: Activity Assay

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: (A) Confluent ECs were incubated with MPO (20 nM) for the indicated times (0-120 min) and cellular levels of eNOS and MPO assessed in cell lysates by Western Blotting. (B, C) Confluent ECs on coverslips were incubated with MPO (20 nM) for 2 h, washed and then fixed with paraformaldehyde and immunostained for MPO (red) or fibronectin (green) by laser confocal fluorescence microscopy. Serial images were taken along the z-plane using the z-stack feature included in the microscopy software. Images were processed using ImageJ, where the apical, intracellular and basolateral planes were determined by referencing the DAPI-stained cell nuclei (blue).

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Incubation, Western Blot, Fluorescence, Microscopy, Software, Staining

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: ECs were incubated in the absence (control, CTL) or presence of MPO (20 nM) for 2 h, washed and then non-treated or treated with H2O2 (50 μM) for 10 min in buffer containing 10 μM L-Arg and 3.3 μCi/mL [3H]-L-Arg. ECs were also stimulated with A23187 (1 μM) for 10 min. (A) eNOS catalytic activity was indexed as the conversion of [3H]-L-Arg into [3H]-L-Citrulline by ECs and expressed as % control values. (B) L-Arg uptake was indexed as the total radiolabel in EC lysates and expressed as % control. (C) EC L-Citrulline levels in (A) were normalized to the corresponding L-Arg uptake levels in (B) and expressed as % control. Data are the mean ± SEM, n=3 independent experiments; *p<0.05, **p<0.01 vs. MPO/H2O2 (one-way ANOVA with Bonferroni’s post-hoc test), ns (non-significant).

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Incubation, Activity Assay

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: (A) ECs were incubated with MPO (20 nM) for 2 h, washed and pre-treated with or without the intracellular calcium-chelator BAPTA-AM (10 μM) for 30 min. Cells were then treated with H2O2 (50 μM) for the indicated times. Cell lysates were assessed for eNOS Ser-1179 phosphorylation or total eNOS by Western Blot. (C) ECs were incubated with MPO (20 nM) for 2 h, washed and replaced with buffer lacking (−Ca2+) or containing (+Ca2+) 1.5 mM calcium prior to the treatment with H2O2 (50 μM) for 2 or 5 mins. Cell lysates were assessed for eNOS Ser-1177 phosphorylation or total eNOS by Western Blot. Note: immunoblots in (A) or (C) of p-eNOS (Ser-1179) and eNOS are from the same gel and nitrocellulose membrane, initially probed for p-eNOS (Ser-1179), stripped, and re-probed for eNOS. (B & D) The band intensities of eNOS phosphorylation at Ser-1179 were quantified by densitometry using ImageJ and expressed as % untreated control ECs. Data are the mean ± SEM from n=3 independent experiments; *p<0.05, **p<0.01 using a two-way ANOVA.

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Incubation, Western Blot, Membrane

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: ECs were incubated in the absence or presence of MPO (20 nM) for 2 h, washed and treated with H2O2 (50 μM) for the indicated times. Cell lysates were analyzed for eNOS phosphorylation status at Ser-1179 or Thr-497 and total eNOS by Western Blotting. Note: the immunoblots for p-eNOS (Ser-1179) and p-eNOS (Thr-497) are derived from the same gel and nitrocellulose membrane, initially probed for p-eNOS (Ser-1179), stripped, and then re-probed for p-eNOS (Thr-497). The eNOS blot shown represents the identical test samples run at the same time on a separate gel and nitrocellulose membrane, which was probed for total eNOS. The band intensities of eNOS phosphorylation at (B) Ser-1179 and (C) Thr-497 were quantified by densitometry using ImageJ and expressed as a % of untreated control ECs or ECs containing MPO alone. Data are mean ± SEM, n=3 independent experiments; ***p<0.001, ****p<0.0001 comparing curves using a two-way ANOVA.

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Incubation, Western Blot, Derivative Assay, Membrane

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: (A) ECs were incubated in the absence or presence of MPO (20 nM) for 2 h, washed and then exposed to varying H2O2 concentrations (0-50 μM) for 5 min. Cell lysates were analyzed for eNOS phosphorylation status at Ser-1179 and Thr-497 or total eNOS by Western Blotting. Note: the immunoblots for p-eNOS (Thr-497) and eNOS are from the same gel and nitrocellulose membrane, initially probed for p-eNOS Thr-497, stripped, and probed for total eNOS. The p-eNOS (Ser-1179) blot shown represents the identical test samples run at the same time on a separate gel and nitrocellulose membrane, which was probed for p-eNOS (Ser-1179). The band intensities of eNOS phosphorylation at (B) Ser-1179 or (C) Thr-497 were quantified by densitometry using ImageJ and expressed as a % of untreated control ECs or ECs containing MPO alone. Data are the mean ± SEM from immunoblots of n=3 independent experiments, *p<0.05, ****p<0.0001 by two-way ANOVA.

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Incubation, Western Blot, Membrane

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: (A, B) ECs were incubated with 20 nM MPO for 2 h, washed and loaded with 5 μM Fura-2-AM for 30 min. Cells were incubated in the absence (Control, CTL) or presence of U73122 (2.5 μM) for 5 min prior to addition of 50 μM H2O2 and calcium imaging. Fluorescent images were taken every 5 s at 510 nm emission with alternating 340/380 nm excitation. Intracellular calcium was quantified by the ratio-metric analysis of F340/F380 images. Data in (A) are representative of n=3 independent experiments and in (B) the mean ± SEM of n=3 independent experiments; *p<0.05. (C-E) ECs were incubated with 20 nM MPO for 2 h, washed and pre-treated with or without U73122 (1 μM) for 5 min before H2O2 (50 μM) treatment for the indicated times. Cell lysates were assessed for eNOS phosphorylation at Ser-1179 or Thr-497 and total eNOS by Western Blotting. Note the immunoblots for p-eNOS (Ser-1179) and eNOS are from the same gel and nitrocellulose membrane that was initially probed for p-eNOS (Ser-1179), stripped, and re-probed for total eNOS. The p-eNOS (Thr-497) blot shown is the identical test samples run at the same time on a separate gel and the nitrocellulose membrane probed for p-eNOS (Thr-497). (D, E) The band intensities of eNOS phosphorylation at Ser-1179 or Thr-497 were quantified by densitometry using ImageJ and expressed as % untreated control ECs or ECs containing MPO alone. Data are the mean ± SEM from immunoblots of n=3 independent experiments; *p<0.05, **p<0.01, ****p<0.0001 using a two-way ANOVA.

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Incubation, Imaging, Western Blot, Membrane

Journal: Free radical biology & medicine

Article Title: Endothelial-Transcytosed Myeloperoxidase Activates Endothelial Nitric Oxide Synthase via a Phospholipase C-Dependent Calcium Signaling Pathway

doi: 10.1016/j.freeradbiomed.2020.12.448

Figure Lengend Snippet: (A) During heightened vascular inflammation increased circulating levels of leukocyte derived MPO are transcytosed through endothelial cells and into the sub-endothelial extracellular matrix (ECM). (B) In the presence of H2O2, endothelial-localized MPO catalyzes the formation of HOCl that (C) impairs the cellular uptake of the eNOS substrate, L-arginine (L-Arg), via the cationic amino acid transporter-1 (CAT-1). In parallel, MPO-derived HOCl also (D) activates phospholipase C (PLC)-dependent increases in intracellular calcium, sourced from (E) the extracellular environment. (F) This increased intracellular calcium activates yet-to-be defined protein kinases/phosphatases that afford increases in eNOS phosphorylation at Ser-1179 and de-phosphorylation at Thr-497, (G) resulting in an increase in eNOS activity and NO production. (H) The MPO-dependent increase in NO may, however, not translate into increased NO bioactivity as MPO in the presence of H2O2 can catalytically consume NO via its NO oxidase activity. Note: this summary scheme does not accurately illustrate the specific sub-cellular locations at which the different oxidative and cell signaling events occur, the nature of which require further investigation.

Article Snippet: Primary antibodies included mouse monoclonal antibodies against eNOS (BD Biosciences) and HOCl-oxidized protein (Clone 2D10G9; that does not cross-react with epitopes generated by oxidative reactions involving nitrating species, transition metals or lipid peroxidation reactions [ 40 ]) or rabbit polyclonal antibodies against MPO (Merck Millipore), tubulin (Sigma), phospho-eNOS (Ser-1177/Ser-1179) (Merck Millipore) and phospho-eNOS (Thr-495/Thr-497) (Merck Millipore).

Techniques: Derivative Assay, De-Phosphorylation Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 1. H2O2-mediated eNOS activation in endothelial cells and rabbit aorta. A, confluent PAECs were equilibrated in PSS and then treated with PSS alone (CTL) or 250 M H2O2 for 20 min prior to the determination of L-[3H]citrulline production as described under “Experimental Procedures.” B, PAECs were incubated for 30 min in PSS containing 200 M IBMX and 200 M L-arginine in the presence or absence of 500 M L-NAME as indicated. Cells were then exposed to buffer alone (CTL) or 250 M H2O2 for 30 min, and cGMP was deter- mined as described under “Experimental Procedures.” Data are pre- sented as the increase in cGMP minus the value obtained for cells treated with L-NAME alone (2.4 0.4 pmol/mg, n 10). C, thoracic aortic rings isolated from New Zealand White rabbits were suspended in organ chambers in the absence or presence of 500 M L-NAME for 30 min. Vessels were then contracted with phenylephrine (1 M), and relaxation was assessed in response to 250 M H2O2. Data represent the mean S.E. of 9–12 independent experiments (p 0.05 versus control (*) or H2O2 (†)).

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activation Assay, Incubation, Isolation, Control

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 2. H2O2-induced changes in eNOS phosphorylation. PAECs were treated with 300 M H2O2 for the indicated times (A) or with increasing concentrations of H2O2 for 30 min (D). After treatment, PAECs were lysed, eNOS was immunoprecipitated, and phosphorylation status at Ser-1177 and Thr-495 was assessed using immunoblotting with phosphorylation-specific antibodies. Anti-eNOS monoclonal antibody was used to detect total eNOS in the immunoprecipitates. Composite densitometric analyses are shown for time-dependent (B and C) and concentration- dependent (E and F) changes in eNOS phosphorylation. Data represent mean S.E. from 3–5 independent experiments. *, p 0.05 versus 0 min or 0 M H2O2.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 4. H2O2 treatment activates Akt in endothelial cells. Post- confluent monolayers of PAECs were treated with 300 M H2O2 for the indicated times (A) or the indicated concentration of H2O2 for 30 min (B). After treatment, cells were lysed, and Akt activation was examined by immunoblotting with an antibody specific for the phosphorylated (Ser-473) form of Akt (p-Akt). Total Akt was assessed using anti-Akt polyclonal antibody. The lower panels represent composite densitomet- ric data (mean S.E.) from four independent experiments. *, p 0.05 versus zero time or 0 M H2O2.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Concentration Assay, Activation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 3. H2O2-induced changes in the eNOS phosphorylation status are reversible. Postconfluent monolayers of PAECs were treated with 250 M H2O2 for 10 min followed by H2O2 removal with three washes using PSS. The cells were then incubated for the indicated times and lysed, eNOS was immunoprecipitated, and phosphorylation status was assessed at Ser-1177 and Thr-495 using immunoblotting with phosphorylation- specific antibodies as in Fig. 2. Results are representative of two independent experiments.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 5. Effect of PI 3-K inhibitors on Akt activation and eNOS phosphoryl- ation status. PAECs were incubated for 30 min in PSS alone or PSS containing LY294002 (LY, 20 M), wortmannin (WM, 200 nM) or catalase (500 IU/ml) prior to treatment with H2O2 (300 M) for a fur- ther 30 min. PAECs were then lysed, and the lysates were analyzed for Akt activa- tion and total Akt (A) by immunoblotting as in Fig. 4. Data are representative of three independent experiments. Lysates were also subjected to eNOS immunopre- cipitation prior to immunoblotting with antibodies specific for eNOS phosphoryl- ated at Ser-1177 (A) or Thr-495 (B). Com- posite densitometry results are also shown for eNOS phosphorylated at Thr- 495 (B) and represent the mean S.E. of three independent experiments. *, p 0.05 versus no H2O2 exposure.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activation Assay, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 6. PI 3-K and H2O2-induced stimulation of eNOS activity. A, PAECs in PSS were incubated in the absence or presence of LY294002 (LY, 20 M) for 30 min. PAECs were then treated with buffer alone or 250 M H2O2 for 20 min prior to the assessment of eNOS catalytic activity as determined by L-[3H]citrulline formation as de- scribed under “Experimental Procedures.” B, PAECs were incubated for 30 min in PSS containing 200 M IBMX and 200 M L-arginine with or without LY294002 (LY, 20 M) for 30 min prior to treatment with 250 M H2O2 or vehicle (CTL). After 30 min, PAEC lysates were analyzed for cGMP as described (23). Data represent the mean S.E. of 6–8 independent experiments and are expressed as a percentage of the values obtained for H2O2 alone. *, p 0.05 versus CTL.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activity Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 7. Inhibition of Akt activation and implications for changes in eNOS phosphorylation. A, PAECs were infected with adenovirus containing a constitutively active form of Akt (Ad.myr.Akt) with a hemagglutinin tag (HA) or control adenovirus expressing LacZ () for 48 h. PAECs were then equilibrated in PSS for 30 min followed by treatment with 300 M H2O2 or vehicle for 20 min. PAECs were then lysed and analyzed for phosphorylated Akt or hemagglutinin-Akt by immunoblotting. Lysates were also immunoprecipitated with anti- eNOS antibody, and the immunoprecipitates were analyzed for total eNOS or eNOS phosphorylated at Ser-1177. B, PAECs were infected with adenovirus containing a dominant-negative form of Akt (Ad.dn.Akt) or control adenovirus expressing LacZ () for 48 h followed by treatment with H2O2 and lysis as in A. PAECs were then analyzed for total or phosphorylated Akt by immunoblotting and total or Ser- 1177 eNOS as in A. C, PAECs were treated as in B except that eNOS phosphorylation was assayed at position Thr-495. Immunoblots shown are representative of three independent experiments.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Infection, Control, Expressing, Western Blot, Immunoprecipitation, Dominant Negative Mutation, Lysis

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 8. Role of Src, ErbB receptor family, and calcium in H2O2- mediated Akt activation and changes in eNOS phosphorylation. PAECs were incubated (30 min) in the absence or presence of PP2 (25 M), AG1478 (50 M), or BAPTA-AM (25 M) for 30 min prior to treatment with 300 M H2O2 for 30 min. A, PAEC lysates were analyzed for Akt activation by immunoblotting using antibodies specific for phos- phorylated or total Akt. PAEC lysates were immunoprecipitated with anti-eNOS antibody, and the immunoprecipitates were examined for eNOS phosphorylation at Ser-1177 (B), Thr-495 phosphorylation (C), or total eNOS as in Fig. 5. Corresponding densitometric data represent the mean S.E. of 4–6 independent experiments (p 0.05 versus H2O2 (*) or no H2O2 (†))

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activation Assay, Phospho-proteomics, Incubation, Western Blot, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

doi: 10.1074/jbc.m109107200

Figure Lengend Snippet: FIG. 9. Role of Src and intracellular Ca2 in H2O2-induced activation of eNOS. A, PAECs in PSS were incubated in the absence or presence of PP2 (25 M; n 8) or BAPTA-AM (20 M; n 3) for 30 min. PAECs were then treated with 250 M H2O2 (20 min) or vehicle alone (CTL) prior to the assessment of eNOS catalytic activity deter- mined by L-[3H]citrulline formation as described under “Experimental Procedures.” B, PAECs were incubated for 30 min in PSS containing 200 M IBMX and 200 M L-arginine with or without PP2 (25 M) or BAPTA-AM (20 M) for 30 min prior to treatment with 250 M H2O2 or vehicle (CTL). After 30 min, PAEC lysates were analyzed for cGMP as described (23). Data represent the mean S.E. of 3–8 independent experiments and are expressed as a percentage of the values obtained for H2O2 alone. *, p 0.05 versus control.

Article Snippet: The polyclonal antibody against phospho-eNOS (Thr-495) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activation Assay, Incubation, Activity Assay, Control