Journal:

Article Title: HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P 3

doi: 10.1172/JCI200418004

Figure Lengend Snippet: HDL induces vasodilation in aortae from rats and mice in an eNOS-dependent manner. (a) Thoracic aortic rings from WKY rats were precontracted with PE (1 × 106 mol/l, arrows), and direct relaxation responses to HDL (0.5 mg/ml) or HDL and L-NAME (50 μmol/l) were evaluated. Shown are representative tracings from one experiment of 16. (b) Cumulative findings (mean ± SEM) for maximal relaxation in response to 0.5 mg/ml HDL in the presence of L-NAME (50 μmol/l), SKF-525A ((SKF, 50 μmol/l), or indomethacin (Indo, 10 μmol/l) (n = 6 each). *P < 0.01 vs. HDL. (c) HUVECs loaded with DAF-2DA were stimulated with 0.5 mg/ml HDL in the absence or presence of L-NAME. Cells were fixed and fluorescence was evaluated under a fluorescence microscope. Shown are representative results (n = 5). (d) Dose response of the vasodilatory effect of HDL (n = 3). (e) Thoracic aortic rings from WT 129/C57BL/6 mice and eNOS–/– mice were precontracted with PE, and direct relaxation responses to HDL (0.5 mg/ml) were measured. Shown are representative tracings from one experiment of six.

Article Snippet: For detection of intracellular NO generation, the NO-sensitive fluorescence dye DAF-2DA (Merck Biosciences, Schwalbach, Germany) was applied as described ( 12 ).

Techniques: Fluorescence, Microscopy

Journal:

Article Title: HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P 3

doi: 10.1172/JCI200418004

Figure Lengend Snippet: HDL induces NO release and vasodilation via Akt-mediated eNOS phosphorylation in endothelial cells and in aortic segments. (a) Left panel: [32P]orthophosphate-labeled HUVECs were stimulated with HDL (0.5 mg/ml) in the presence or absence of LY294002 (10 μmol/l). Immunoprecipitated eNOS (ip) was analyzed by autoradiography (n = 3), and amounts of immunoprecipitated protein were detected by Western blotting. Phosphorylation of Akt at Ser473 was determined in cell lysates with a phosphospecific antibody (n = 5). Right panel: Time-dependence of HDL-induced eNOS and Akt phosphorylation at Ser1177 and Ser473, respectively, as analyzed by densitometry (n = 3). (b) Following precontraction of thoracic aortic rings from WKY rats with PE (1 × 10–6 mol/l, arrows), direct relaxation responses to HDL (0.5 mg/ml) in the absence or presence of LY294002 (10 μmol/l) were evaluated. Shown are original tracings from one experiment of eight. (c) Aortic segments perfused with 0.5 mg/ml HDL were fixed and immunostained for phospho-Ser1177-eNOS. Arrows indicate phospho-eNOS staining in the endothelial lining (original magnification, ×200). (d) Fura2-AM–loaded HUVECs were stimulated with 1 mg/ml HDL in the presence or absence of BAPTA-2AM (20 μmol/l) or Ni2+ (5 mM). [Ca2+]i was measured by fluorescence spectroscopy. Original tracings from representative experiments were superimposed for comparison. (e) HUVECs loaded with DAF-2DA and preincubated with BAPTA-2AM (20 μmol/l) or Ni2+ (5 mmol/l) were stimulated with HDL (1 mg/ml) and observed under a fluorescence microscope. Shown are representative results for one experiment of three.

Article Snippet: For detection of intracellular NO generation, the NO-sensitive fluorescence dye DAF-2DA (Merck Biosciences, Schwalbach, Germany) was applied as described ( 12 ).

Techniques: Labeling, Immunoprecipitation, Autoradiography, Western Blot, Staining, Fluorescence, Spectroscopy, Microscopy

Journal:

Article Title: HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P 3

doi: 10.1172/JCI200418004

Figure Lengend Snippet: Lysophospholipid signaling mediates HDL-, SPC-, S1P-, and LSF-induced Akt and eNOS phosphorylation as well as [Ca2+]i increase. (a) HUVECs were stimulated with HDL (0.5 mg/ml) or with 10 μmol/l each (left) or 0.5–5 μmol/l each (right) SPC, S1P, and LSF, with or without preincubation with 100 ng/ml PTX for 16 hours. “PTX control” indicates PTX treatment alone. Cell lysates were analyzed for phospho–Ser473-Akt (p-Ser473-Akt) and phospho–Ser1177-eNOS by Western blotting. Loading controls for total eNOS and total Akt content are shown. All results are representative of one experiment of three. (b) HUVECs loaded with DAF-2DA were stimulated with SPC, S1P, and LSF (10 μmol/l each) and observed under a fluorescence microscope. Shown are representative results for one experiment of three. (c) Fura2-AM–loaded HUVECs were stimulated with SPC, S1P, and LSF (10 μmol/l each), and [Ca2+]i was measured by fluorescence spectroscopy. Original tracings from representative experiments were superimposed for comparison. (d) Concentration dependence of [Ca2+]i increase in HUVECs stimulated with SPC, S1P, and LSF measured as described in c.

Article Snippet: For detection of intracellular NO generation, the NO-sensitive fluorescence dye DAF-2DA (Merck Biosciences, Schwalbach, Germany) was applied as described ( 12 ).

Techniques: Western Blot, Fluorescence, Microscopy, Spectroscopy, Concentration Assay