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.